Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

中文翻译：

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工程化细胞质乙酰辅酶A合成将产油酵母红冬孢酵母中的脂质生产与氮饥饿解耦

产油酵母能够积累非常高水平的中性脂质，特别是在碳和氮限制过量的条件下（具有高C/N比的培养基）。这使得有必要使用两步过程来实现高水平的生物量和脂质。为了简化这一过程，通过建立一种胞质乙酰辅酶A形成途径替代ATP-柠檬酸裂合酶催化的途径，将脂质合成与氮饥饿解偶联可能是有用的。在这项工作中，我们通过过表达编码同源磷酸酮醇酶 (Xfpk) 和异源磷酸转乙酰酶 (Pta) 的基因，引入了阿佐里红冬孢子虫中乙酰辅酶 A (AcCoA) 形成的新细胞质途径。当在含有不同C/N比率的培养基中培养时，工程菌株PTAPK4表现出比野生型菌株更高的脂质含量并产生更高的脂质浓度。在对葡萄糖/木糖混合物进行的生物反应器过程中，为了模拟从木质纤维素材料生产脂质的工业过程，我们发现与野生型相比，工程菌株的最终脂质浓度增加了 89%。这表明转化菌株可以产生比野生型更高的细胞生物量和高脂质含量。转化菌株还证明了在执行该过程中相对于野生型的优势，脂质产量分别为0.13和0.05。我们的结果表明，R. azoricum 中同源 Xfpk 和异源 Pta 活性的过度表达创造了一种新的胞质 AcCoA 供应，可将脂质产生与氮饥饿解耦。这种代谢修饰可以提高培养条件下的脂质产量，适合使用木质纤维素水解产物开发工业生物过程。