Center for the Utilization of Biological Engineering in Space, Berkeley, California, USA.
Department of Bioengineering, University of California, Berkeley, California, USA.
Biotechnol Bioeng. 2024 Jan;121(1):139-156. doi: 10.1002/bit.28532. Epub 2023 Aug 28.
Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two-component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, nonstress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Due to uncertainty in the two-component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single-stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single-stage continuous systems. This work expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.
铜绿假单胞菌属的细菌以能够大量产生聚-β-羟基丁酸（PHB）而闻名，这使它们成为生物塑料生产的有吸引力的候选者。PHB 的天然合成发生在代谢应激期间，而这些生物体中调节 PHB 积累起始的过程尚未完全理解。筛选铜绿假单胞菌 4G11 的 RB-TnSeq 转座子文库使我们能够鉴定出两个明显的、未被表征的双组分系统基因，当从基因组中删除这两个基因时，它们能够在平衡、非应激生长条件下提高 PHB 的生产力。我们观察到，在单独从基因组中删除每个基因时，PHB 的生产力相对于野生型亲本菌株平均增加了 56%和 41%。然而，在无氮不平衡生长条件下，增加的 PHB 表型消失，这表明该表型是特定于快速生长、充足、非应激生长的。生物生产模型表明，这种表型可能是由于突变菌株中对氮限制诱导的代谢应激的依赖降低，从而启动 PHB 生产。由于双组分系统输入信号和调控子的不确定性，这些基因赋予这种表型的机制仍不清楚。由于这种不确定性，在这种配置下，与分批系统相比，这种类似的产物产率可以使用单级连续生物反应器系统，而无需使用以前使用的许多 PHB 生物生产方案，这种表型的菌株可能允许使用单级连续生物反应器系统。生物生产模型表明，在使用单级连续系统时，与野生型生物相比，在细胞中省略这种调节可能会使 PHB 生产力提高 24%。这项工作扩展了我们对铜绿假单胞菌中 PHB 积累调节的理解，特别是在过渡到不平衡生长状态时启动这一过程的调节。
