Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA ; Present address: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Systems Biotechnology, Forschungszentrum Juelich, 52425 Juelich, Germany.
Biotechnol Biofuels. 2015 Feb 26;8:31. doi: 10.1186/s13068-015-0209-z. eCollection 2015.
Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations.
An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L(-1) glycerol, produced 2.3 g L(-1) of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g(-1) of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L(-1)) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L(-1) glycerol, 40 g L(-1) glucose, and 40 g L(-1) xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L(-1) of TAGs, representing 51.2% of the CDM. In addition, when 20 g L(-1) glycerol was pulse-loaded into the culture with 40 g L(-1) glucose and 40 g L(-1) xylose at the stationary growth phase, MITGM-173 produced 14.3 g L(-1) of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L(-1) glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g(-1) on the initial addition and 0.279 g g(-1) on the pulse addition of glycerol.
We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.
可再生燃料生产过程中产生的甘油，作为发酵生产附加值材料的潜在基质具有吸引力。产油菌 Rhodococcus opacus 的工程菌株 MITXM-61 在高浓度葡萄糖和木糖的培养基上大量生产细胞内三酰基甘油(TAGs)，用于生产基于脂质的生物燃料。然而，在甘油培养基上，MITXM-61 不产生 TAGs，生长状况不佳。本研究的目的是构建一种能够在高浓度甘油下进行高密度细胞培养的产 TAGs 的 Rhodococcus opacus 菌株。
采用适应性进化策略提高了 Rhodococcus opacus MITXM-61 中甘油向 TAGs 的转化率。在含有 16 g/L 甘油的限定培养基中生长的进化株 MITGM-173 产生了 2.3 g/L 的 TAGs，占细胞干重 (CDM) 的 40.4%，每消耗 1g 甘油产生 0.144 g TAG。MITGM-173 能够在高浓度(大于 150 g/L)的甘油中生长。在初始浓度为 20 g/L 甘油、40 g/L 葡萄糖和 40 g/L 木糖的培养基中培养时，MITGM-173 能够同时消耗混合基质，并产生 13.6 g/L 的 TAGs，占 CDM 的 51.2%。此外，当在静止生长阶段用 40 g/L 葡萄糖和 40 g/L 木糖脉冲加载 20 g/L 甘油时，MITGM-173 产生了 14.3 g/L 的 TAGs，占 CDW 的 51.1%，尽管在培养物中仍观察到残留的甘油。在葡萄糖/木糖混合物中添加 20 g/L 甘油，在初始添加和甘油脉冲添加时，每消耗 1g 甘油的 TAG 产率分别为 0.170 g/g 和 0.279 g/g。
与亲本菌株相比，我们生成了一株产 TAG 的 Rhodococcus opacus MITGM-173 菌株，该菌株在甘油利用方面有显著提高。本研究表明，进化后的 Rhodococcus opacus 菌株在利用混合糖原料生产先进可再生燃料方面具有很大的潜力，同时补充甘油。
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