초록
<P><B>ABSTRACT</B></P><P>To balance the flux of an engineered metabolic pathway to achieve high yield of target product is a major challenge in metabolic engineering. In previous work, the collaborative regulation of CO<SUB>2</SUB> transport and fixation was investigated with co‐overexpressing exogenous genes regulating both CO<SUB>2</SUB> transport (<I>sbtA</I> and <I>bicA</I>) and PEP carboxylation (phosphoenolpyruvate (PEP) carboxylase (<I>ppc</I>) and carboxykinase (<I>pck</I>)) under trc promoter in <I>Escherichia coli</I> for succinate biosynthesis. For balancing metabolic flux to maximize succinate titer, a combinatorial optimization strategy to fine‐tuning CO<SUB>2</SUB> transport and fixation process was implemented by promoter engineering in this study. Firstly, based on the energy matrix a synthetic promoter library containing 20 rationally designed promoters with strengths ranging from 0.8% to 100% compared with the widely used trc promoter was generated. Evaluations of <I>rfp</I> and <I>cat</I> reporter genes provided evidence that the synthetic promoters were stably and had certain applicability. Secondly, four designed promoters with different strengths were used for combinatorial assembly of single CO<SUB>2</SUB> transport gene (<I>sbtA</I> or <I>bicA</I>) and single CO<SUB>2</SUB> fixation gene (<I>ppc</I> or <I>pck</I>) expression. Three combinations, such as Tang1519 (P<SUB>4</SUB>‐<I>bicA </I>+ pP<SUB>19</SUB>‐<I>pck</I>), Tang1522 (P<SUB>4</SUB>‐<I>sbtA </I>+ P<SUB>4</SUB>‐<I>ppc</I>), Tang1523 (P<SUB>4</SUB>‐<I>sbtA </I>+ P<SUB>17</SUB>‐<I>ppc</I>) with a more than 10% increase in succinate production were screened in bioreactor. Finally, based on the above results, co‐expression of the four transport and fixation genes were further investigated. Co‐expression of <I>sbtA</I>, <I>bicA</I>, and <I>ppc</I> with weak promoter P<SUB>4</SUB> and <I>pck</I> with strong promoter P<SUB>19</SUB> (AFP111/pT‐P<SUB>4</SUB>‐<I>bicA</I>‐P<SUB>4</SUB>‐<I>sbtA </I>+ pACYC‐P<SUB>19</SUB>‐<I>pck</I>‐P<SUB>4</SUB>‐<I>ppc</I>) provided the best succinate production among all the combinations. The highest succinate production of 89.4 g/L was 37.5% higher than that obtained with empty vector control. This work significantly enhanced succinate production through combinatorial optimization of CO<SUB>2</SUB> transport and fixation. The promoter engineering and combinatorial optimization strategies used herein represents a powerful approach to tailor‐making metabolic pathways for the production of other industrially important chemicals. Biotechnol. Bioeng. 2016;113: 1531–1541. © 2016 Wiley Periodicals, Inc.</P>