Background Acyl-(acyl carrier protein (ACP)) reductase (AAR) is a key enzyme for hydrocarbon biosynthesis in cyanobacteria, reducing fatty acyl-ACPs to aldehydes, which are then converted into hydrocarbons by aldehyde-deformylating oxygenase (ADO). Previously, we compared AARs from various cyanobacteria and found that hydrocarbon yield in Escherichia coli coexpressing AAR and ADO was highest for AAR from Synechococcus elongatus PCC 7942 (7942AAR), which has high substrate affinity for 18-carbon fatty acyl-ACP, resulting in production of mainly heptadecene. In contrast, the hydrocarbon yield was lowest for AAR from Synechococcus sp. PCC 7336 (7336AAR), which has a high specificity for 16-carbon substrates, leading to production of mainly pentadecane. However, even the most productive AAR (7942AAR) still showed low activity; thus, residues within AAR that are nonconserved, but may still be important in hydrocarbon production need to be identified to engineer enzymes with improved hydrocarbon yields. Moreover, AAR mutants that favor shorter alkane production will be useful for producing diesel fuels with decreased freezing temperatures. Here, we aimed to identify such residues and design a highly productive and specific enzyme for hydrocarbon biosynthesis in E. coli. Results We introduced single amino acid substitutions into the least productive AAR (7336AAR) to make its amino acid sequence similar to that of the most productive enzyme (7942AAR). From the analysis of 41 mutants, we identified 6 mutations that increased either the activity or amount of soluble AAR, leading to a hydrocarbon yield improvement in E. coli coexpressing ADO. Moreover, by combining these mutations, we successfully created 7336AAR mutants with ~ 70-fold increased hydrocarbon production, especially for pentadecane, when compared with that of wild-type 7336AAR. Strikingly, the hydrocarbon yield was higher in the multiple mutants of 7336AAR than in 7942AAR. Conclusions We successfully designed AAR mutants that, when coexpressed with ADO in E. coli, are more highly effective in hydrocarbon production, especially for pentadecane, than wild-type AARs. Our results provide a series of highly productive AARs with different substrate specificities, enabling the production of a variety of hydrocarbons in E. coli that may be used as biofuels.

中文翻译：

---

通过工程蓝藻酰基-（酰基载体蛋白）还原酶提高碳氢化合物的产量。

背景酰基-（酰基载体蛋白（ACP））还原酶（AAR）是蓝藻生物合成碳氢化合物的关键酶，将脂肪酰基-ACPs还原为醛，然后通过醛脱甲酰加氧酶（ADO）将其转化为碳氢化合物。之前，我们比较了来自各种蓝藻的 AAR，发现共表达 AAR 和 ADO 的大肠杆菌中烃类产量最高的是来自细长聚球藻 PCC 7942 (7942AAR) 的 AAR，它对 18 碳脂肪酰基-ACP 具有高底物亲和力，从而产生产量主要是十七碳烯。相比之下，来自 Synechococcus sp. 的 AAR 的烃产率最低。PCC 7336 (7336AAR)，对 16 碳底物具有高特异性，主要生产十五烷。然而，即使是最高效的 AAR (7942AAR) 仍然表现出低活性；因此，需要鉴定 AAR 中非保守但在烃生产中仍可能很重要的残基，以设计提高烃产量的酶。此外，有利于较短烷烃生产的 AAR 突变体可用于生产冷冻温度降低的柴油燃料。在这里，我们旨在识别这些残留物并设计一种用于大肠杆菌中碳氢化合物生物合成的高产和特异性酶。结果 我们将单个氨基酸取代引入到最低产的 AAR (7336AAR) 中，使其氨基酸序列与最高产的酶 (7942AAR) 的氨基酸序列相似。通过对 41 个突变体的分析，我们确定了 6 个突变，它们增加了可溶性 AAR 的活性或数量，从而提高了共表达 ADO 的大肠杆菌的碳氢化合物产量。此外，通过结合这些突变，与野生型 7336AAR 相比，我们成功地创建了 7336AAR 突变体，其碳氢化合物产量增加了约 70 倍，尤其是十五烷。引人注目的是，7336AAR 的多个突变体中的烃产率高于 7942AAR。结论 我们成功设计了 AAR 突变体，当在大肠杆菌中与 ADO 共表达时，与野生型 AAR 相比，其在烃生产中更有效，尤其是对十五烷的生产。我们的结果提供了一系列具有不同底物特异性的高产 AAR，从而能够在大肠杆菌中生产各种可用作生物燃料的碳氢化合物。7336AAR多个突变体的烃产率高于7942AAR。结论 我们成功设计了 AAR 突变体，当在大肠杆菌中与 ADO 共表达时，与野生型 AAR 相比，其在烃生产中更有效，尤其是对十五烷的生产。我们的结果提供了一系列具有不同底物特异性的高产 AAR，从而能够在大肠杆菌中生产各种可用作生物燃料的碳氢化合物。7336AAR多个突变体的烃产率高于7942AAR。结论 我们成功设计了 AAR 突变体，当在大肠杆菌中与 ADO 共表达时，与野生型 AAR 相比，其在烃生产中更有效，尤其是对十五烷的生产。我们的结果提供了一系列具有不同底物特异性的高产 AAR，从而能够在大肠杆菌中生产各种可用作生物燃料的碳氢化合物。