3.3 Engineering ZM4 with the self-flocculating phenotype
To elucidate the functions of genes involved in the biosynthesis and
degradation of c-di-GMP, we targeted genes encoding proteins with PDE
activities for c-di-GMP degradation to explore the effect of their
combinatory knockout on the development of the self-flocculating
phenotype in ZM4.
When both ZMO1055 and ZMO0401 were deleted, the flocculation efficiency
of the bacterial cells improved to 24.4%, and the flocculation
efficiency was further improved to 34.5% for the double knockout mutant
with both ZMO1055 and ZMO1487 deleted (Fig. 5A). No further improvement
in the self-flocculating phenotype was observed when all three genes
were deleted in ZM4. However, such genetic manipulation is preferred for
reducing the genome of Z. mobilis to engineer this species as a
more reliable method to accommodate heterogeneous genes more
effectively.
Cellulose fibrils have been validated as the chemical basis for
developing the self-flocculating phenotype in ZM401(Xia et al., 2018).
ZM4 also contains a bacterial cellulose synthase (bcs ) operon
composed of ZMO1082, ZMO1083, ZMO1084, and ZMO1085. ZMO1082 was
predicted to be a putative gene encoding a short peptide composed of 67
amino acid residues only (Xia et al., 2018), which is less likely to be
functional and thus can be manipulated together with ZMO1083. Therefore,
we engineered ZM4 with the overexpression of ZMO1082-1083 and
ZMO1082-1084 and the whole bcs operon ZMO1082-1085 to investigate
their contribution to the self-flocculation of the bacterial cells. As a
result, the development of the self-flocculating phenotype was observed
in the mutants with their flocculating efficiencies of 28.9%, 43.1%
and 66.7%, respectively (Fig. 5A).
When both strategies were employed in ZM4, enhancing its intracellular
accumulation of c-di-GMP to 94.42 pg/mg protein through the deletion of
ZMO1055, ZMO0401, and ZMO1487, and the biosynthesis of cellulose fibrils
through the overexpression of the bcs operon, a flocculating
efficiency of 97.3% was observed for the bacterial cells, which was
higher than that of 92.5% detected with ZM401 (Fig. 5A). Morphologies
were further shown for ZM4 strains engineered with the deletion of
ZMO1055, ZMO0401, and ZMO1487, overexpression of the bcs operon,
and the combination of these two strategies (Fig. 5B).
Fig. 5
When industrial strains are
engineered with new phenotypes, such as the self-flocculation of
microbial cells for more advantages, their production performance should
not be compromised. Z. mobilis is ethanologenic, and suitable for
producing cellulosic ethanol. Therefore, we compared ethanol
fermentation performance between the strain engineered with the
self-flocculating phenotype and its unicellular wild-type ZM4. As can be
seen (Fig. S1), no difference was observed when medium supplemented with
100 g/L glucose, equivalent to
total sugars in the hydrolysate of lignocellulosic biomass, was
fermented to produce ethanol.