초록
<P><B>Abstract</B></P> <P>Rubisco-based engineered <I>Escherichia coli</I> MZLFB (<I>E. coli</I> BL21(DE3) Δ<I>zwf</I>, Δ<I>ldh</I>, Δ<I>frd</I>) containing heterologous phosphoribulokinase (Prk) and Ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) was constructed for the mixotrophic growth. However, <I>in situ</I> CO<SUB>2</SUB> recycling was hindered by clogs of pyruvate during glucose metabolism, which consequently resulted in an insufficient regeneration of NAD<SUP>+</SUP> through the <I>pflB</I>-mediated ethanol production. Recombinant plasmid pLOI295 (encodes pyruvate decarboxylase and alcohol dehydrogenase II, referred to as the Pdc-based carbon tap valve (CTV) for convenience) was introduced into <I>E. coli</I> MZLFB + CTV to bypass the <I>pflB</I>-mediated ethanol production. Results show that while the C-2/C-1 ratio (<I>i.e.,</I> the molar ratio of ethanol and acetate to formate and total CO<SUB>2</SUB>) for parental strain MZLFB was 1.0 ± 0.1, the C-2/C-1 for MZLFB + CTV increased to 1.6 ± 0.1. This indicates that the Pdc-based CTV enhanced the performance of <I>in situ</I> CO<SUB>2</SUB> recycling. By simultaneously utilizing glucose and CO<SUB>2</SUB>, the fermentation product yield of MZLFB + CTV exceeded the normal theoretical yield and reached 2.2 ± 0.0 (mol/mol). <I>In silico</I> analysis shows that 61% of the glucose consumption went through the Rubisco-based engineered pathway when the CTV was equipped. Also shown are the average CO<SUB>2</SUB> consumption rate of 55.3 mg L<SUP>−1</SUP>·h<SUP>−1</SUP> and an average ethanol production rate of 144.8 mg L<SUP>−1</SUP>·h<SUP>−1</SUP>. The conversion of CO<SUB>2</SUB> to ethanol through the Rubisco-based engineered pathway and the Pdc-based carbon tap valve is important for mixotrophic growth, since these two modules serve as the energy sink to achieve intracellular energy balance. Also, during mixotrophic growth, ATP production from a certain percentage (39% in this study) of the EMP pathway activity is needed for mixotrophic growth.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Substantial mixotrophic growth can be achieved by draining pyruvate through pLOI295. </LI> <LI> The forced conversion of pyruvate to ethanol leads to a new redox balance. </LI> <LI> Mixotrophic growth reached the fermentation product yield reached 2.2 ± 0.0. </LI> <LI> <I>In silico</I> analysis shows that the average CO<SUB>2</SUB> consumption was 55.3 mg L<SUP>−1</SUP>·h<SUP>−1</SUP>. </LI> <LI> The doubling time of mixotrophic <I>E. coli</I> was 5.9 ± 0.5 h. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>