Abstract
BackgroundAlthough 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.ResultsIn 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0392-4) contains supplementary material, which is available to authorized users.
Highlights
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
When samples were cultured with glucose, the expression of glycerol kinase (GK) kept decreasing to reach an extremely low level during the fatty acid accumulation stage (K, L, M)
The expression of G3PD1 (NAD+) was increased more than fivefold, and the expression of the other glycerol-3-phosphate dehydrogenase (G3PD) was not significantly downregulated (Fig. 2a) after nitrogen exhaustion (Fig. 2b). These results were consistent with the previously performed transcriptome analysis, and indicated that the G3PDs may play an important role during fatty acid accumulation in M. alpina [18]
Summary
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. In pursuit of a higher commercial added-value to relieve the pressure for cost-efficiency, the use of glycerol to produce polyunsaturated fatty acids (PUFAs) has gathered increasing interest in recent years [6]. When using glycerol as carbon source, the key issue is the relatively low assimilation efficiency that limits downstream metabolic processes. This is presumably due to the insufficient coordination of the enzymes involved in the primary metabolic steps of glycerol assimilation [7, 10, 11]. Glycerol is catabolized by glycerol kinase (GK, EC 2.7.1.30) to glycerol-3-phosphate, which can be used either as a precursor for lipid biosynthesis or converted by glycerol-3-phosphate dehydrogenase (G3PD, Hao et al Microb Cell Fact (2015) 14:205
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