Hao Guangfei, Chen Haiqin, Gu Zhennan, Zhang Hao, Chen Wei, Chen Yong Q
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People's Republic of China.
Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, People's Republic of China.
Microb Cell Fact. 2015 Dec 23;14:205. doi: 10.1186/s12934-015-0392-4.
Although some microorganisms can convert glycerol into valuable products such as polyunsaturated fatty acids, the yields are relative low due primarily to an inefficient assimilation of glycerol. Mortierella alpina is an oleaginous fungus which preferentially uses glucose over glycerol as the carbon source for fatty acid synthesis.
In the present study, we metabolically engineered M. alpina to increase the utilization of glycerol. Glycerol kinase and glycerol-3-phosphate dehydrogenase control the first two steps of glycerol decomposition. GK overexpression increased the total fatty acid content by 35%, whereas G3PD1, G3PD2 and G3PD3 had no significant effect. Overexpression of malic enzyme (ME1) but not glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase significantly increased fatty acid content when glycerol was used as carbon source. Simultaneous overexpression of GK and ME1 enabled M. alpina to accumulate fatty acids efficiently, with a 44% increase in fatty acid content (% of dry weight), a 57% increase in glycerol to fatty acid yield (g/g glycerol) and an 81% increase in fatty acid production (g/L culture). A repeated batch process was applied to relieve the inhibitory effect of raw glycerol on arachidonic acid synthesis, and under these conditions, the yield reached 52.2 ± 1.9 mg/g.
This study suggested that GK is a rate-limiting step in glycerol assimilation in M. alpina. Another restricting factor for fatty acid accumulation was the supply of cytosolic NADPH. We reported a bioengineering strategy by improving the upstream assimilation and NADPH supply, for oleaginous fungi to efficiently accumulate fatty acid with glycerol as carbon source.
尽管一些微生物能够将甘油转化为诸如多不饱和脂肪酸等有价值的产物，但产量相对较低，主要原因是甘油的同化效率低下。高山被孢霉是一种产油真菌，在脂肪酸合成过程中，它优先利用葡萄糖而非甘油作为碳源。
在本研究中，我们对高山被孢霉进行代谢工程改造以提高甘油的利用率。甘油激酶和甘油-3-磷酸脱氢酶控制甘油分解的前两个步骤。甘油激酶的过表达使总脂肪酸含量增加了35%，而甘油-3-磷酸脱氢酶1、甘油-3-磷酸脱氢酶2和甘油-3-磷酸脱氢酶3没有显著影响。当以甘油作为碳源时，苹果酸酶（ME1）的过表达显著增加了脂肪酸含量，而葡萄糖-6-磷酸脱氢酶、6-磷酸葡萄糖酸脱氢酶或异柠檬酸脱氢酶的过表达则没有这种效果。甘油激酶和苹果酸酶的同时过表达使高山被孢霉能够高效积累脂肪酸，脂肪酸含量（占干重的百分比）增加了44%，甘油到脂肪酸的产量（克/克甘油）增加了57%，脂肪酸产量（克/升培养物）增加了81%。采用重复分批培养工艺以减轻粗甘油对花生四烯酸合成的抑制作用，在此条件下，产量达到52.2±1.9毫克/克。
本研究表明甘油激酶是高山被孢霉中甘油同化的限速步骤。脂肪酸积累的另一个限制因素是胞质NADPH的供应。我们报道了一种通过改善上游同化和NADPH供应来使产油真菌以甘油作为碳源高效积累脂肪酸的生物工程策略。
