A phosphate-controlled autolysis system for PHA recovery from

Cupriavidus necator H16

Alejandro Salinas1,2,3, Callum McGregor1, Victor Irorere1, Marisin Tenorio4, Nigel P. Minton1,
Mara Cea2,3, Katalin Kovács1,5
1 BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, The University of Nottingham, Nottingham, NG7 2RD, UK
2 Chemical Engineering Department, Universidad de La Frontera, Temuco, Chile
3 Scientific and Technological Bioresources Nucleus BIOREN-UFRO, Universidad de La Frontera, Temuco, Chile
4 Doctoral program in Sciences of Natural Resources, Universidad de La Frontera, Temuco, Chile
5 School of Pharmacy, University Park, University of Nottingham, Nottingham NG7 2RD, UK

Cell lysis in PHA-producing strains C. necator

Introduction Growth and production decoupling using PpstS
H16 and C. necator H16 ΔmtgA

Cupriavidus necator H16 is a metabolically versatile bacterium
that, when grown under limited nutrient conditions, such as
phosphate and nitrogen limitation, accumulates large amounts
of intracellular polyhydroxyalkanoates (PHAs).
Rational engineering of C. necator H16 requires the use of
appropriate promoters to regulate gene expression. Despite
significant progress in broadening the repertoire of both
constitutive and inducible promoters for C. necator H16, these
are frequently incompatible with industrial scale-up. Auto-
inducible promoters have emerged as a promising alternative
for big scale fermentation. Phosphate starvation promoters, in
particular, are attractive as they tend to be tightly regulated
and show a strong induction response.
C. necator H16 was grown using different concentrations of
initial inorganic phosphate (Pi) to check if the length of the
growth and production phases could be controlled by a simple
change in media composition (Fig 2).
A B
The use of the autolysis system was then studied in the PHA-
producing strains, C. necator H16 wild type and C. necator
H16 ΔmtgA (Fig 4). MtgA is a non-essential monofunctional
glycosyltransferase involved in peptidoglycan synthesis [2]
and its deletion in Escherichia coli improved PHA
accumulation by an enlargement of cell size [3].
CFU dropped by more than 95% after 21 and 18 h when C.
necator H16 and C. necator H16 ΔmtgA carried the autolysis
system. However, PHA accumulation was very low.
A B

Intracellular products such as PHA require efficient release

from the cells. PHA recovery methods are often too costly or
not environmentally friendly. Cell autolysis systems have
emerged as a an alternative for sustainable recovery of PHA.
Fig 2. Effect of initial phosphate concentration on cell growth and induction of the
PpstS promoter. Growth (A) and normalized fluorescence (B) curves of C. necator
H16 ΔphaCAB harbouring the PpstS-rfp construct.

In this study, we identified and characterized two promoters,

PpstS and PphoA, which are associated with C. necator H16
phosphate starvation response. We then showed that PpstS
can be used to decouple growth and production in a controlled
manner modifying the initial phosphate concentration. Finally,
PpstS was used to design a lysozyme-based two-step autolysis
system for PHA recovery from C. necator H16 and the mutant
C. necator H16 ΔmtgA.
Identification of phosphate limitation-inducible
promoters
C. necator H16 genome was analysed and two putative
operons potentially involved in its phosphate starvation
response were identified (Fig 1, A).
The identified promoters were cloned immediately upstream of
a monomeric red fluorescent protein gene (rfp) (Fig 1, B) and
their induction under phosphate-limiting conditions validated
using C. necator H16 ΔphaCAB (Fig 1, C-D).
Design of a lysozyme-based autolysis system
for PHA recovery
A lysozyme-based autolysis system triggered by phosphate
limitation was designed to easily release the PHA granules
from the cells.
The autolysis system requires periplasmic expression of
lysozyme C from Gallus gallus to destabilize the cell
membrane (Fig 3, A) [1]. Considering this, lysC was cloned
upstream of PpstS using different signal peptides and the
growth of C. necator H16 ΔphaCAB carrying these constructs
studied (Fig 3, B-D) .
Fig 4. Cell lysis response of the PHA-producing strains C. necator H16 and C.
necator H16 ΔmtgA. (A) Schematic representation of the peptidoglycan-
synthesising activity of the non-essential monofunctional glycosyltransferase MtgA.
OM: outer-membrane, IM: inner-membrane. (B) Growth curves of C. necator H16
and C. necator H16 ΔmtgA harbouring the cell lysis construct. WT: C. necator H16,
ΔmtgA: C. necator H16 ΔmtgA.
A two-step system for PHA production and
recovery
A two-step system, which includes a fully dedicated step for
biomass and polymer build-up, and a second step for cell
lysis, was designed. Nitrogen limitation was used to promote
PHA accumulation and avoid early cell-lysis, while phosphate-
free medium was used to trigger cell lysis.
Ethylenediaminetetraacetic acid (EDTA) supplementation
during the lysis step was also assessed as it can enhance cell
lysis in some gram-negative bacteria [1],[4].

A B

C D

WT pPpstS-RFP WT pPpstS-SPPpOprF-lysC

Fig 1. Identification and characterization of the phosphate starvation inducible
promoters PpstS and PphoA from C. necator H16. (A) Putative operons involved in the
phosphate starvation response of C. necator H16. (B) Schematic representation of
the reporting constructs. Normalized fluorescence curves of C. necator H16
ΔphaCAB harbouring the reporter constructs. Cultivations were carried out using
either phosphate-rich medium (C) or phosphate-limited (D) medium.
Fig 3. Design of a lysozyme-based cell lysis system inducible by phosphate
limitation for C. necator H16. (A) Enzymatic hydrolysis of peptidoglycan by
lysozyme C (LysC). NAM: N-acetylmuramic acid, NAG: N-acetylglucosamine, OM:
outer-membrane, IM: inner-membrane. (B) Schematic representation of the cell
lysis constructs. Growth curves of C. necator H16 ΔphaCAB harbouring the cell
lysis constructs using 5 mM (C) and 0.5 mM (D) initial phosphate concentration.
Fig 5. A two-step system for PHA production and extraction using C. necator H16
and C. necator H16 ΔmtgA. Lysis efficiencies using the two-step system (A). Cells
were grown for 48 h using nitrogen limited medium to allow PHA accumulation. The
cultures were washed and resuspended in phosphate-free medium and cultured for
an additional 24 h with and without 1 mM EDTA. Example of PHA release after the
use of the two-step cell lysis system (B). WT: C. necator H16, ΔmtgA: C. necator
H16 ΔmtgA.

Acknowledgements
Conclusions
This work was supported by
BBSRC/EPSRC grant award ▪ Two C. necator H16 promoters inducible by phosphate limitation were identified and characterized.
BB/L0139404/1 and the
▪ A system to decouple growth and production based on the initial phosphate concentration was designed using PpstS.
FONDECYT postdoctoral
grant #3200748. ▪ A two-step lysozyme-based autolysis system triggered by phosphate limitation was designed for PHA production and
recovery.

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