Abstract
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 Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) 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 T. oleaginosus 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.
Highlights
Biofuels such as biodiesel continue to be favorites as renewable transportation fuels, since their use requires little modification to current engines
With the amount of NADPH provided through the phosphate pathway (PPP) limited, the metabolic stoichiometry would allow glucose conversion to TAG through the pyruvate dehydrogenase (PDH) and PDH bypass routes with yields 0.275 and 0.289 g/g, respectively
With the expression of the PDH bypass, our aim was to enhance yeast TAG production compared to the control strain of T. oleaginosus, which has only the native PDH pathway with ACL to generate cytosolic acetyl-CoA
Summary
Biofuels such as biodiesel continue to be favorites as renewable transportation fuels, since their use requires little modification to current engines. Biodiesel and renewable diesel can be produced from renewable biological sources such as vegetable oils and animal fats. They are biodegradable, non-toxic, and have a low emission profile. Due to the increased demand for biodiesel and the limited and/or undesirable sources of biodiesel raw materials such as rape seed oil, soy bean oil, or palm oil, it is of importance to expand biodiesel raw materials to non-food materials like microbes. The benefits of using microbes for production of oils are that they are affected neither by seasons nor by climates, they are able to produce high lipid contents, and the oils can be produced from a wide variety of sources with short production times (Adrio, 2017). We show how triacylglycerol (TAG) yield in the oleaginous yeast Trichosporon oleaginosus (formerly Cryptococcus curvatus) can be improved by modifying central metabolic pathways
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