Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan.
Appl Microbiol Biotechnol. 2014 Mar;98(6):2453-60. doi: 10.1007/s00253-013-5401-0. Epub 2013 Dec 13.
Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (ΔpflA, Δpta, ΔackA, ΔpoxB, Δdld, and a dual mutant; ΔpflA + Δdld) and their parent strain, BW25113, were grown on 20 g l(-1) xylose for P(LA-co-3HB) production. The single deletions of ΔpflA, Δpta, and Δdld increased the LA fraction (58-66 mol%) compared to BW25113 (56 mol%). In particular, the ΔpflA + Δdld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in ΔpflA, Δpta, and Δdld mutants, and BW25113. The ΔpflA + gatC strain achieved a productivity of 8.3 g l(-1), which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l(-1) xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l(-1). On the other hand, the ΔpflA + Δdld strain grown on 30 g l(-1) xylose synthesized 6.4 g l(-1) P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000-114,000.
聚（丙交酯-共-3-羟基丁酸酯）（P（LA-co-3HB））以前是在工程大肠杆菌中从木糖生产的。本研究的目的是通过两种代谢工程方法提高聚合物的生产力和 P（LA-co-3HB）中的 LA 分数：（1）消除乳酸生成的竞争途径的缺失，（2）过表达半乳糖醇转运蛋白（GatC），这有助于 ATP 非依赖性木糖摄取。工程大肠杆菌突变体（ΔpflA、Δpta、ΔackA、ΔpoxB、Δdld 和双突变体；ΔpflA+Δdld）及其亲本 BW25113 在 20 g l(-1)木糖上生长以生产 P（LA-co-3HB）。与 BW25113（56 mol%）相比，单个缺失ΔpflA、Δpta 和Δdld 增加了 LA 分数（58-66 mol%）。特别是，ΔpflA+Δdld 菌株产生的 P（LA-co-3HB）含有 73 mol% LA。此外，GatC 的过表达增加了 ΔpflA、Δpta 和Δdld 突变体以及 BW25113 中聚合物的产率和 LA 分数。ΔpflA+gatC 菌株的生产力达到 8.3 g l(-1)，这是理论最大产率的 72%。因此，为了消除碳源的限制，添加了更高浓度的木糖。结果，携带 gatC 的 BW25113 在 40 g l(-1)木糖上生长，达到了 14.4 g l(-1)的最高 P（LA-co-3HB）生产力。另一方面，在 30 g l(-1)木糖上生长的ΔpflA+Δdld 菌株合成了 6.4 g l(-1)的 P（LA-co-3HB），同时保持了最高的 LA 分数（73 mol%）。结果表明 GatC 可用于增强木糖生产 P（LA-co-3HB），基因缺失可提高 P（LA-co-3HB）中的 LA 分数。获得的聚合物的重均分子量在 34000-114000 范围内。
