With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an important barrier to traditional metabolic engineering,however,the identification of ratelimiting step(s)for the improvement of specific cellular functions is often difficult.Meanwhile,in the case of synthetic biology,more and more BioBricks could be constructed for targeted purposes,but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge.Owing to the lack of steady-state kinetic data for overall flux,balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials.A strategy called targeted engineering is proposed in an effort to solve this problem.Briefly,a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors,substrates and each enzyme will be analyzed systematically.Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays.To demonstrate its practical application,biosynthesis pathways for the production of important products,e.g.chemicals,nutraceuticals and drug precursors,have been engineered in Escherichia coli and Saccharomyces cerevisiae.These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.
Mortierella alpina has been considered as the most effective producer of arachidonic acid(ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successfully, as they activated the desaturation system on the endoplasmic reticulum. Additionally, in M. alpina the ARA exists in several forms, such as triacylglycerol(TAG), and diacylglycerol(DAG). These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil. However, few works revealed detailed fatty acid distribution among lipid classes, which to some extent impeded the accurate regulation in ARA accumulation. Herein, this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M. alpina. The result demonstrates that TAG was the dominant component of the total lipids, and it is the main form for ARA storage. The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady. Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modi fication of saturated and monounsaturated fatty acids in other lipid classes. This work could be helpful for further optimization of ARA-rich TAG production.
