Enhancing single-cell hyaluronic acid biosynthesis by microbial morphology engineering

Abstract

Microbial morphology engineering is a novel approach for cell factory to improve the titer of target product in bio-manufacture. Hyaluronic acid (HA), a valuable glycosaminoglycan polymerized by HA synthase (HAS), a membrane protein, is particularly selected as the model product to improve its single-cell HA-producing capacity via morphology engineering. DivIVA and FtsZ, the cell-elongation and cell division related protein, respectively, were both down/up dual regulated in C. glutamicum via weak promoter substitution or plasmid overexpression. Different from the natural short-rod shape, varied morphologies of engineered cells, i.e. small-ellipsoid-like (DivIVA-reduced), bulb-like (DivIVA-enhanced), long-rod (FtsZ-reduced) and dumbbell-like (FtsZ-enhanced), were observed. Applying these morphology-changed cells as hosts for HA production, the reduced expression of both DivIVA and FtsZ seriously inhibited normal cell growth; meanwhile, overexpression of DivIVA didn't show morphology changes, but overexpression of FtsZ surprisingly change the cell-shape into long and thick rod with remarkably enlarged single-cell surface area (more than 5.2-fold-increase). And finally, the single-cell HA-producing capacity of the FtsZ-overexpressed C. glutamicum was immensely improved by 13.5-folds. Flow cytometry analyses verified that the single-cell HAS amount on membrane was enhanced by 2.1 folds. This work is pretty valuable for high titer synthesis of diverse metabolic products with microbial cell factory.

Keywords: DivIVA protein; Down/up dual regulation; Enlarged cells; FtsZ protein; Morphology engineering; Single-cell HA-Producing capacity.

Fig. 1

Morphology engineering design of DivIVA and FtsZ to obtain enlarged cells with enhanced surface-area. (a) The locating positions of DivIVA and FtsZ in a cell. (b) Deduced morphology shifts of C. glutamicum after reducing or enhancing expression of DivIVA and FtsZ. (c) Morphology-regulated cells would improve the HAS expression and HA synthesis due to the enhanced surface-area.

Fig. 2

Down or up expression strategies of divIVA and ftsZ. (a,b) Down-regulated strain: C. glutamicum-P_divIVA: P_lac and C. glutamicum-P_ftsZ: P_lac, respectively, constructed by in situ promoter replacement through single crossover homologous recombination. (c,d) Up-regulated strain: C. glutamicum/pEC-DivIVA and C. glutamicum/pEC-FtsZ, respectively, constructed by plasmids overexpression.

Fig. 3

Relative expression of divIVA and ftsZ in C. glutamicum after down/up-regulation of DivIVA and FtsZ with control strain (wild-type) by qRT-PCR. Relative expression levels were measured in triplicate at 24 h by qRT-PCR, and the error bars are the standard deviation. Control: wild-type; DivIVA reduced: C. glutamicum-P_divIVA: P_lac; DivIVA enhanced: C. glutamicum/pEC-DivIVA; FtsZ reduced: C. glutamicum-P_ftsZ: P_lac; FtsZ enhanced: C. glutamicum/pEC-FtsZ.

Fig. 4

Cell morphology changes of the C. glutamicum cells before and after down/up-regulation of DivIVA and FtsZ. Cell morphology were observed at 24 h/48 h by optical microscope, and at 48 h by TEM. Original cell: wild-type; DivIVA reduced expression: C. glutamicum-P_divIVA: P_lac; DivIVA enhanced expression: C. glutamicum/pEC-DivIVA; FtsZ reduced expression: C. glutamicum-P_ftsZ: P_lac; FtsZ enhanced expression: C. glutamicum/pEC-FtsZ.

Fig. 5

Cell morphology and HA biosynthesis with the morphology-changed engineered C. glutamicum strains. Cell morphology were observed at 24 h/48 h by optical microscope, and at 48 h by TEM. OD600 were measured in triplicate from 8 h to 48 h. HA titers were measured in triplicate at 24 h and 48 h and the error bars are the standard deviation. Original cell: wild-type; DivIVA reduced expression: C. glutamicum-P_divIVA: P_lac/pX-AB; DivIVA enhanced expression: C. glutamicum/pEC-AB-DivIVA; FtsZ reduced expression: C. glutamicum-P_ftsZ: P_lac/pX-AB; FtsZ enhanced expression: C. glutamicum/pEC-AB-FtsZ.

Fig. 6

HAS synthesis in single cell indirectly determined by flow cytometry. A: wild-type; B: C. glutamicum/pEC-RFP:AB; C: C. glutamicum/pEC-RFP:AB-FtsZ. The bacteria were cultured in the fermentation medium for 24 h, then diluted to 10⁶/mL with deionized water for flow cytometry. The fluorescence less than 100 was considered as background. The abscissa represented the fluorescence value; the ordinate represented the number of cells. The white line represented the average fluorescence intensity.