Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Microbial Chemistry, Department of Chemistry - Ångström, Uppsala University, Box 523, SE-75120, Uppsala, Sweden.
Sci Rep. 2020 Mar 11;10(1):4515. doi: 10.1038/s41598-020-61100-4.
The integrative aspect on carbon fixation and lipid production is firstly implemented in cyanobacterium Synechocystis sp. PCC 6803 using metabolic engineering approach. Genes related to Calvin-Benson-Bassham (CBB) cycle including rbcLXS and glpD and free fatty acid recycling including aas encoding acyl-ACP synthetase were practically manipulated in single, double and triple overexpressions via single homologous recombination. The significantly increased growth rate and intracellular pigment contents were evident in glpD-overexpressing (OG) strain among all strains studied under normal growth condition. The triple aas_glpD_rbcLXS-overexpressing (OAGR) strain notably gave the highest contents of both intracellular lipids and extracellular free fatty acids (FFAs) of about 35.9 and 9.6% w/DCW, respectively, when compared to other strains at day 5 of cultivation. However, the highest intracellular lipid titer and production rate were observed in OA strain at day 5 (228.7 mg/L and 45.7 mg/L/day, respectively) and OG strain at day 10 (358.3 mg/L and 35.8 mg/L/day, respectively) due to their higher growth. For fatty acid (FA) compositions, the main saturated fatty acid of palmitic acid (C16:0) was dominantly found in both intracellular lipid and secreted FFAs fractions. Notably, intracellular FA proportion of myristic acid (C14:0) was induced in all engineered strains whereas the increase of stearic acid (C18:0) composition was found in extracellular FFAs fraction. Altogether, these overexpressing strains efficiently produced higher lipid production via homeostasis balance on both its lipid synthesis and FFAs secretion.
利用代谢工程方法，首先在集胞藻 PCC 6803 中实现了碳固定和脂质生产的综合方面。通过同源重组，在单个、双个和三个过表达中实际操作了与卡尔文-本森-巴斯汉姆（CBB）循环相关的基因，包括 rbcLXS 和 glpD 以及游离脂肪酸回收，包括编码酰基辅酶 A 合成酶的 aas 基因。在正常生长条件下，所有研究菌株中，glpD 过表达（OG）菌株的增长率和细胞内色素含量显著增加。与其他菌株相比，三重 aas_glpD_rbcLXS 过表达（OAGR）菌株在培养第 5 天分别具有最高的细胞内脂质和细胞外游离脂肪酸（FFAs）含量，约为 35.9%和 9.6%w/DCW。然而，OA 菌株在第 5 天（分别为 228.7mg/L 和 45.7mg/L/天）和 OG 菌株在第 10 天（分别为 358.3mg/L 和 35.8mg/L/天）具有最高的细胞内脂质滴度和生产速率，这是由于它们的生长速度较快。对于脂肪酸（FA）组成，棕榈酸（C16:0）的主要饱和脂肪酸主要存在于细胞内脂质和分泌的 FFAs 馏分中。值得注意的是，所有工程菌株的细胞内 FA 豆蔻酸（C14:0）比例都有所增加，而细胞外 FFAs 馏分中的硬脂酸（C18:0）组成增加。总之，这些过表达菌株通过其脂质合成和 FFAs 分泌的平衡有效地产生了更高的脂质产量。
