Abstract
Various organic acids have huge potential as industrial
platform chemicals. Biotechnological routes of organic
acid production are currently being sought, so that
fossil resources and petrochemistry could be replaced
with renewable resources. Microbial production of organic
acids imposes stresses on the organism and understanding
the physiology of micro-organisms which have been
genetically engineered to produce an organic acid, can
make valuable contributions to the development of
production organisms for biorefineries.
Production of D-xylonate, an industrial platform chemical
with high application potential, was successfully
demonstrated in various yeast species. D-xylonate is
produced from D-xylose via D-xylono-?-lactone that can be
hydrolysed to D-xylonate spontaneously or with the aid of
a lactonase enzyme. Various ways to improve production of
D-xylonate in the yeast Saccharomyces cerevisiae,
Kluyveromyces lactis or Pichia kudriavzevii as production
organisms were successfully applied. The best D-xylonate
production was obtained by expression of the D-xylose
dehydrogenase encoding gene xylB from Caulobacter
crescentus and the highest D-xylonate titre was achieved
with P. kudriavzevii that produced 171 and 146 g
D-xylonate l-1, at a rate of 1.4 or 1.2 g l-1 h-1, at pH
5.5 and pH 3, respectively.
The consequences of D-xylonate production on the
physiology of S. cerevisiae were studied in detail, both
at population and single-cell level. D-xylonate and
D-xylono-?-lactone were produced and also exported from
the cells from the very start of cultivation in D-xylose,
even in the presence of D-glucose. There was no apparent
preference for export of either compound. However, great
amounts of D-xylono-?-lactone and/or D-xylonate was
accumulated inside the cells during the production.
The D-xylonolactone lactonase encoding gene xylC was
co-expressed with the D-xylose dehydrogenase encoding
gene xylB (both genes from C. crescentus). This lead to a
significant increase in the D-xylonate production rate
compared to cells expressing only xylB and showed that
accumulation of D-xylonate and protons releases during
hydrolysis, was harmful for the cells. The accumulation
of D-xylonate led to lost vitality and acidification of
the cytosol, as determined by loss of pHluorin (a pH
dependent fluorescent protein) fluorescence. This loss of
fluorescence was faster in cells co-expressing xylC with
xylB compared to cells expressing xylB alone. The
decrease in vitality and challenges in export of
D-xylonate are major obstacles for D-xylonate production
by S. cerevisiae. The excellent D-xylonate producer, P.
kudriavzevii also accumulated large amounts of D-xylonate
and suffered decreased vitality, especially when
D-xylonate was produced at low pH.
The stress response to weak organic acids is highly
dependent on the properties of the acids and the presence
of high concentrations of weak organic acids may lead to
lost viability. The role of Pdr12, a membrane
transporter, in resistance to weak organic acids was
studied and found to be highly dependent on the acid.
Deletion of PDR12 led to improved tolerance to formic and
acetic acids, a feature that makes this modification
interesting for micro-organisms used in biorefining of
lignocellulosic hydrolysates that commonly contain these
acids.
Biotechnological production of D-xylonic acid with yeast
clearly has the potential of becoming an industrially
applicable process. In order for biotechnological
production processes to become economically feasible,
biorefinery approaches in which lignocellulosic
hydrolysates or other biomass side- or waste streams are
used as raw materials need to be employed. This thesis
provides new understanding on how production of an
organic acid affects the production host and presents
novel approaches for studying and increasing the
production.
Original language | English |
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Qualification | Doctor Degree |
Awarding Institution |
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Supervisors/Advisors |
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Award date | 6 Jun 2014 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 978-951-38-8147-4 |
Electronic ISBNs | 978-951-38- 8148-1 |
Publication status | Published - 2014 |
MoE publication type | G5 Doctoral dissertation (article) |
Keywords
- yeast
- D-xylonate
- metabolic engineering
- organic acids
- stress responses
- cytosolic pH
- Pdr12
- D-xylose