Environmental Regulation of PndbA600, an Auto-Inducible Promoter for Two-Stage Industrial Biotechnology in Cyanobacteria.
Environmental Regulation of PndbA600, an Auto-Inducible Promoter for Two-Stage Industrial Biotechnology in Cyanobacteria.
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DOI：
10.3389/fbioe.2020.619055
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发表时间：
2020
影响因子：
5.7
通讯作者：
Amtmann A
中科院分区：
文献类型：
--
作者：
Madsen MA;Hamilton G;Herzyk P;Amtmann A
蓝细菌是一种光合作用的原核生物，作为可持续发展的平台，利用可再生资源（光，水和空气）在能源，食品，环境和医学方面的各种应用。尽管蓝细菌为工业生物技术提供了诱人的前景，但缓慢的生长速率在通常需要大量生物质并且通常对细胞有毒的过程中构成了重大挑战。两阶段培养策略是一种有吸引力的解决方案，通过将生物质积累（阶段I）和工业过程（阶段II）解耦来防止任何不期望的生长抑制。在蓝藻，两个阶段的战略涉及昂贵的转移方法之间的阶段I和II，很少有工作集中在使用不同的生长和静止期的分批培养自动调节阶段过渡。在本研究中，我们确定并表征了生长阶段特异性启动子，它可以作为一个自动诱导开关，以调节两个阶段的生物过程中的蓝藻。首先，从比较集胞藻PCC 6803的两个生长阶段和六种营养条件的新RNAseq数据集中鉴定了生长阶段特异性基因，包括低Mg和低K的两个新转录组。当培养物从指数生长过渡到稳定期生长时，II型NADH脱氢酶（ndBA）显示出稳健的诱导。然后，在聚球藻属PCC 7002中表达PndbA 600：GFP之后，详细表征600-bp启动子序列（PndbA 600）的行为。培养密度和生长培养基的分析表明，PndbA 600激活不依赖于增加本身的文化密度，但对N的可用性和另一个激活因子存在于静止期培养物（因子X）的废培养基。PndbA 600失活依赖于培养密度的变化以及N可用性或因子X的变化。电子传递抑制研究表明，光合作用的活性PndbA 600水平的具体增强。我们的研究结果总结在一个模型中描述的PndbA 600，它现在可以通知的两个阶段的工业过程中蓝藻的合理设计的环境调节。
Cyanobacteria are photosynthetic prokaryotes being developed as sustainable platforms that use renewable resources (light, water, and air) for diverse applications in energy, food, environment, and medicine. Despite the attractive promise that cyanobacteria offer to industrial biotechnology, slow growth rates pose a major challenge in processes which typically require large amounts of biomass and are often toxic to the cells. Two-stage cultivation strategies are an attractive solution to prevent any undesired growth inhibition by de-coupling biomass accumulation (stage I) and the industrial process (stage II). In cyanobacteria, two-stage strategies involve costly transfer methods between stages I and II, and little work has been focussed on using the distinct growth and stationary phases of batch cultures to autoregulate stage transition. In the present study, we identified and characterised a growth phase-specific promoter, which can serve as an auto-inducible switch to regulate two-stage bioprocesses in cyanobacteria. First, growth phase-specific genes were identified from a new RNAseq dataset comparing two growth phases and six nutrient conditions in Synechocystis sp. PCC 6803, including two new transcriptomes for low Mg and low K. A type II NADH dehydrogenase (ndbA) showed robust induction when the cultures transitioned from exponential to stationary phase growth. Behaviour of a 600-bp promoter sequence (PndbA600) was then characterised in detail following the expression of PndbA600:GFP in Synechococcus sp. PCC 7002. Culture density and growth media analyses showed that PndbA600 activation was not dependent on increases in culture density per se but on N availability and on another activating factor present in the spent media of stationary phase cultures (Factor X). PndbA600 deactivation was dependent on the changes in culture density and in either N availability or Factor X. Electron transport inhibition studies revealed a photosynthesis-specific enhancement of active PndbA600 levels. Our findings are summarised in a model describing the environmental regulation of PndbA600, which can now inform the rational design of two-stage industrial processes in cyanobacteria.