Cyanobacteria, a group of photosynthetic prokaryotes, are being increasingly explored for direct conversion of carbon dioxide to useful chemicals. However, efforts to engineer these photoautotrophs have resulted in low product titers. This may be ascribed to the bottlenecks in metabolic pathways, which need to be identified for rational engineering. We engineered the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801 to produce succinate, an important platform chemical. Previously, engineering of the model cyanobacterium S. elongatus PCC 7942 has resulted in succinate titer of 0.43 g l−1 in 8 days. Building on the previous report, expression of α-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and phosphoenolpyruvate carboxylase yielded a succinate titer of 0.6 g l−1 in 5 days suggesting that PCC 11801 is better suited as host for production. Profiling of the engineered strains for 57 intermediate metabolites, a number of enzymes and qualitative analysis of key transcripts revealed potential flux control points. Based on this, we evaluated the effects of overexpression of sedoheptulose-1,7-bisphosphatase, citrate synthase and succinate transporters and knockout of succinate dehydrogenase and glycogen synthase A. The final construct with seven genes overexpressed and two genes knocked out resulted in photoautotrophic production of 0.93 g l−1 succinate in 5 days. While the fast-growing strain PCC 11801 yielded a much higher titer than the model strain, the efficient photoautotrophy of this novel isolate needs to be harnessed further for the production of desired chemicals. Engineered strains of S. elongatus PCC 11801 showed dramatic alterations in the levels of several metabolites suggesting far reaching effects of pathway engineering. Attempts to overexpress enzymes deemed to be flux controlling led to the emergence of other potential rate-limiting steps. Thus, this process of debottlenecking of the pathway needs to be repeated several times to obtain a significantly superior succinate titer.

中文翻译：

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快速生长蓝藻细长聚球藻 PCC 11801 的代谢工程，用于光合自养生产琥珀酸。

蓝细菌是一组光合原核生物，正在越来越多地探索将二氧化碳直接转化为有用的化学物质。然而，设计这些光合自养生物的努力导致产品滴度低。这可能归因于代谢途径中的瓶颈，需要确定这些瓶颈以进行合理的工程设计。我们设计了最近报道的、快速生长和健壮的蓝藻，细长聚球藻 PCC 11801，以生产琥珀酸盐，一种重要的平台化学品。此前，模型蓝藻 S. elongatus PCC 7942 的工程已在 8 天内产生 0.43 g l-1 的琥珀酸滴度。在先前报告的基础上，α-酮戊二酸脱羧酶、琥珀酸半醛脱氢酶和磷酸烯醇丙酮酸羧化酶的表达产生的琥珀酸滴度为 0。5 天内 6 g l-1 表明 PCC 11801 更适合作为生产宿主。对 57 种中间代谢物的工程菌株进行分析、多种酶和关键转录物的定性分析揭示了潜在的通量控制点。基于此，我们评估了sedoheptulose-1,7-bisphosphatase、柠檬酸合酶和琥珀酸转运蛋白的过表达以及琥珀酸脱氢酶和糖原合酶A的敲除的影响。具有七个基因过表达和两个基因敲除的最终构建体导致光合自养生产0.93 g l-1 琥珀酸盐在 5 天内。虽然快速生长的菌株 PCC 11801 产生的滴度比模型菌株高得多，但需要进一步利用这种新型分离物的有效光自养来生产所需的化学品。S的工程菌株。elongatus PCC 11801 显示出几种代谢物水平的显着变化，表明途径工程的深远影响。过度表达被认为是通量控制的酶的尝试导致了其他潜在的限速步骤的出现。因此，该途径的消除瓶颈过程需要重复多次以获得显着优越的琥珀酸滴度。