Roche Christine M, Glass N Louise, Blanch Harvey W, Clark Douglas S
The Chemical and Biomolecular Engineering Department, The University of California, Berkeley, California, 94720; The Energy Biosciences Institute, The University of California, Berkeley, California, 94720.
Biotechnol Bioeng. 2014 Jun;111(6):1097-107. doi: 10.1002/bit.25211. Epub 2014 Apr 3.
Microbially produced triacylglycerol (TAG) is a potential feedstock for the production of biodiesel, but its commercialization will require high yields from low-cost renewable feedstocks such as lignocellulose. The present study employs a multi-gene approach to increasing TAG biosynthesis in the filamentous fungus Neurospora crassa. We demonstrate the redirection of carbon flux from glycogen biosynthesis towards fatty acid biosynthesis in a glycogen synthase deletion strain (Δgsy-1). Furthermore, combining Δgsy-1 with an enhanced TAG biosynthetic strain (acyl-Coenzyme A synthase; Δacs-3) of N. crassa yielded a twofold increase in total fatty acid accumulation over the control strain. The cellulose degrading potential of this double deletion strain was improved by deleting of the carbon catabolite regulation transcription factor (Δcre-1) to create the triple deletion strain Δacs-3 Δcre-1; Δgsy-1. This strain exhibited early and increased cellulase expression, as well as fourfold increased total fatty acid accumulation over the control on inhibitor-free model cellulose medium. The Δcre-1 mutation, however, was not beneficial for total fatty acid accumulation from pretreated lignocellulose. Conversion of dilute-acid pretreated Miscanthus to TAG was maximum in the constructed strain Δacs-3; Δgsy-1, which accumulated 2.3-fold more total fatty acid than the wild-type control strain, corresponding to a total fatty acid yield of 37.9 mg/g dry untreated Miscanthus.
微生物产生的三酰甘油（TAG）是生产生物柴油的潜在原料，但其商业化需要从木质纤维素等低成本可再生原料中获得高产率。本研究采用多基因方法来提高丝状真菌粗糙脉孢菌中TAG的生物合成。我们证明了在糖原合酶缺失菌株（Δgsy-1）中，碳通量从糖原生物合成重定向到脂肪酸生物合成。此外，将Δgsy-1与粗糙脉孢菌的增强型TAG生物合成菌株（酰基辅酶A合成酶；Δacs-3）相结合，使总脂肪酸积累量比对照菌株增加了两倍。通过缺失碳分解代谢物调节转录因子（Δcre-1）来创建三缺失菌株Δacs-3 Δcre-1；Δgsy-1，提高了这种双缺失菌株的纤维素降解潜力。该菌株在无抑制剂的模型纤维素培养基上表现出早期且增强的纤维素酶表达，以及总脂肪酸积累量比对照增加了四倍。然而，Δcre-1突变对于从预处理的木质纤维素中积累总脂肪酸并无益处。在构建的菌株Δacs-3；Δgsy-1中，稀酸预处理芒草向TAG的转化量最大，该菌株积累的总脂肪酸比野生型对照菌株多2.3倍，相当于总脂肪酸产量为37.9mg/g干未处理芒草。
