Pan Zhiwei, Zhu Tao, Domagalski Nathan, Khan Saleem, Koepsel Richard R, Domach Michael M, Ataai Mohammad M
Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Biotechnol Prog. 2006 Sep-Oct;22(5):1451-5. doi: 10.1021/bp060049u.
Our prior work has shown that a pyk mutant of Bacillus subtilis exhibited diminished acidic byproduct accumulation, dramatically elevated phosphoenolpyruvate (PEP) pool, and reduced growth rate. To determine if a low acetate-producing but fast-growing strain of B. subtilis could be developed, we placed the expression of the pyk gene under the control of an inducible promoter. Enzyme measurements proved that PYK activity of the inducible PYK mutant (iPYK) increases with the isopropyl-beta-d-thiogalactopyranoside concentration. Batch growth experiments showed that growth rate and acid formation are closely related to the induction level of pyk. Measurements of cell growth rate and acetate formation of the iPYK mutant at different induction levels revealed that a PYK activity of about 12% of wild-type allows for good growth rate (0.4 h(-)(1) versus 0.63 h(-)(1) of wild-type) and low acetate production (0.26 g/L versus 1.05 g/L of wild-type). This is the first report to our knowledge of a metabolically engineered B. subtilis strain that allows good growth rate and low acid production in batch cultures. Finally, it was found that, by varying the pyk induction level, intracellular PEP concentration can be controlled over a wide range. The intracellular PEP concentration is intimately connected to the regulation of the transport of phosphotransferase system (PTS) sugars in the presence of glucose. Because there is no other method for modulating intracellular PEP levels, this finding represents a major advance in one's ability to dissect the function of the PTS and sugar metabolism in bacteria.
我们之前的研究表明，枯草芽孢杆菌的pyk突变体表现出酸性副产物积累减少、磷酸烯醇丙酮酸（PEP）库显著升高以及生长速率降低。为了确定是否可以构建出一种低产乙酸但生长快速的枯草芽孢杆菌菌株，我们将pyk基因的表达置于可诱导启动子的控制之下。酶活性测定证明，可诱导的PYK突变体（iPYK）的PYK活性随异丙基-β-D-硫代半乳糖苷浓度的增加而升高。分批培养生长实验表明，生长速率和酸形成与pyk的诱导水平密切相关。对不同诱导水平下iPYK突变体的细胞生长速率和乙酸形成的测定结果显示，当PYK活性约为野生型的12%时，可实现良好的生长速率（0.4 h⁻¹，而野生型为0.63 h⁻¹）和较低的乙酸产量（0.26 g/L，而野生型为1.05 g/L）。据我们所知，这是关于一种代谢工程改造的枯草芽孢杆菌菌株的首次报道，该菌株在分批培养中能够实现良好的生长速率和低酸产量。最后，研究发现，通过改变pyk的诱导水平，可以在很宽的范围内控制细胞内PEP的浓度。在葡萄糖存在的情况下，细胞内PEP浓度与磷酸转移酶系统（PTS）糖类转运的调节密切相关。由于没有其他调节细胞内PEP水平的方法，这一发现代表了人们在剖析细菌中PTS功能和糖代谢方面能力的重大进展。
