Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.
Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.
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DOI：
10.1128/spectrum.02149-22
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发表时间：
2022-12-21
影响因子：
3.7
通讯作者：
Han, Jing
中科院分区：
文献类型：
--
作者：
Yang, Haibo;Chen, Junyu;Mitra, Ruchira;Xue, Qiong;Xiang, Hua;Han, Jing
虽然在盐古菌中使用多个复制起点进行染色体复制已被广泛表征，但是否有可能通过基因工程操纵染色体拷贝数尚不清楚，并且它如何影响细胞功能也知之甚少。在这里，我们证明，删除三个活跃的染色体起源在地中海盐藻显着降低其DNA量和倍性数。因此，突变株H. mediacetasei Δ123对紫外线和丝裂霉素C更敏感。令人惊讶的是，细胞大小增加了21.2%，并且摇瓶培养物中的聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV）产量在ΔEPSΔ123中从7.23 g/L提高到8.11 g/L，尽管细胞生长也有所下降。在该突变体中，染色体拷贝数减少，而编码pha 300的巨质粒拷贝数增加。此外，我们的转录组分析显示，参与初级代谢的基因在ΔEPSΔ123中显著下调，而负责淀粉利用和PHBV单体前体供应的基因上调。这表明更多的能量和碳流从初级代谢重定向到PHBV合成，从而增强其PHBV积累。因此，这些研究结果可能提供一个合理的设计，以提高PHBV的合成，通过简单地调整复制起点，以调节染色体/megaplastic拷贝数比，并随后影响细胞代谢和生理功能。重要性盐古菌Haloferax medicinei是一种潜在的PHBV（100%可生物降解塑料）生产商，其碳源廉价。我们以前报道过H. mediaweii具有三个活跃的染色体起点，当这些起点被删除时，一个休眠的起点被激活以启动染色体的复制。在这种情况下，在本研究中，我们首先发现了复制启动和PHBV积累之间的密切联系。我们描述了菌株H的潜在工业优势。medicellacei ΔEPSΔ123，其中包括细胞体积增大21.2%和PHBV产量增加11.2%。我们进一步揭示了导致ΔEPSΔ123菌株中PHBV产量增加的可能机制。总的来说，我们在这里提供了一个概念性的进展，在合成生物学领域通过调节染色体复制，以提高生物基化学品的生产。
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.
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影响因子：
5.9
作者：
Lin L;Chen J;Mitra R;Gao Q;Cheng F;Xu T;Zuo Z;Xiang H;Han J
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嗜盐古菌中受调控的多倍体。
影响因子：
3.7
作者：
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影响因子：
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代谢多功能嗜盐古菌 Haloferax mediterranei（一种聚（3-羟基丁酸酯-co-3-羟基戊酸酯）生产者）的完整基因组序列
影响因子：
3.2
作者：
Han, Jing;Zhang, Fan;Xiang, Hua
通讯作者：
Xiang, Hua