Wang Jia, Butler Robert R, Wu Fan, Pombert Jean-François, Kilbane John J, Stark Benjamin C
Department of Biology, Illinois Institute of Technology, Chicago IL, United States of America.
PLoS One. 2017 Jan 6;12(1):e0168833. doi: 10.1371/journal.pone.0168833. eCollection 2017.
In previous work from our laboratories a synthetic gene encoding a peptide ("Sulpeptide 1" or "S1") with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells' overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.
在我们实验室之前的工作中，设计了一种编码具有高比例甲硫氨酸和半胱氨酸残基的肽（“硫代肽1”或“S1”）的合成基因，使其作为硫库，并将其插入到红平红球菌IGTS8的dsz（脱硫）操纵子中。在本文所述的工作中，将该构建体（dszAS1BC）和克隆到载体pRESX中并受（红球菌）kstD启动子控制的完整dsz操纵子（dszABC）转化到轻红球菌脱硫阴性菌株CW25中。将所得菌株（CW25[pRESX-dszABC]和CW25[pRESX-dszAS1BC]）在以二苯并噻吩（DBT）作为唯一硫源的基本培养基中，通过对数期重复传代（最多100次）进行适应性选择。对于这两种菌株，DBT代谢在选择过程早期达到峰值，然后下降，最终平均为初始转化细胞的四倍；CW25[pRESX-dszAS1BC]达到的最大比活性超过了CW25[pRESX-dszABC]。生长速率分别提高了7倍（CW25[pRESX-dszABC]）和13倍（CW25[pRESX-dszAS1BC]），并且这些提高是稳定的。CW25[pRESX-dszAS1BC]的适应性与质粒拷贝数相比初始转化细胞增加了3 - 5倍相关；全基因组测序表明，在其选择过程中，参与硫代谢、应激反应或DNA甲基化的任何dsz、S1或其他基因及启动子均未发生突变，并且与细胞整体对半胱氨酸和甲硫氨酸的需求相比，S1产生的硫库的影响可能非常小。然而，使用硫库的基因工程与通过适应性选择提高Dsz能力相结合可能是提高生物脱硫能力的可行策略。
