Koivuranta Kari, Castillo Sandra, Jouhten Paula, Ruohonen Laura, Penttilä Merja, Wiebe Marilyn G
VTT Technical Research Centre of Finland Ltd., Espoo, Finland.
Front Microbiol. 2018 Jun 21;9:1337. doi: 10.3389/fmicb.2018.01337. eCollection 2018.
Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast , we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. (formerly ) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.
线粒体丙酮酸脱氢酶（PDH）在产油酵母的脂质生产中起着重要作用，但其他酵母可能会绕过线粒体（PDH旁路），将细胞质中的丙酮酸转化为乙醛，然后再转化为乙酸和乙酰辅酶A，后者进一步转化为脂质。利用基于产油酵母的代谢模型，我们发现将此旁路引入产油酵母中应能提高底物上三酰甘油（TAG）的产量。（以前）是一种产油酵母，它可以利用葡萄糖和木糖生产TAG。基于已测序的基因组，它缺乏完成PDH旁路所需的至少一种酶，即乙醛脱氢酶（ALD），并且在生产条件下可能还缺乏丙酮酸脱羧酶和乙酰辅酶A合成酶。我们将这些基因以各种组合引入到中，并证明在葡萄糖和木糖上TAG的产量都有所提高，特别是在高C/N比的情况下。与PDH旁路一起表达磷脂：二酰基转移酶编码基因进一步提高了脂质产量。工程菌株中木糖上TAG的产量（0.27 g/g）接近理论最大产量0.289 g/g。有趣的是，在一些仅给予部分旁路途径的菌株中，与对照相比TAG产量也有所提高，这表明这些基因可能有助于细胞质乙酰辅酶A的替代途径。代谢模型表明，PDH旁路中底物上TAG产量的提高依赖于ALD产生的NADPH。否则，脂质合成所需的NADPH主要由磷酸戊糖途径（PPP）提供。这将有助于在引入PDH旁路时，与从葡萄糖中观察到的情况相比，从木糖中生产TAG有更大的提高，因为木糖通过PPP的非氧化部分进入代谢。工程菌株中木糖上TAG的产量（0.21 - 0.27 g/g）与从葡萄糖中获得的产量相当，是迄今为止报道的从木糖生产脂质或TAG的最高产量。
