Thesis (DSc (Microbiology))--University of Stellenbosch, 2005. / The technological advancement of modern human civilisation has, until recently, depended
on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over
the last few decades, greater efforts have been made to economise on the use of these nonrenewable
energy resources, and to reduce the environmental pollution caused by their
consumption. In a quest for new sources of energy that will be compatible with a more
sustainable world economy, increased emphasis has been place on researching and
developing alternative sources of energy that are renewable and safer for the environment.
Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately
two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm
sustainability of fuel ethanol as an energy source has prompted consideration for the
use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the
continued availability of fuel ethanol as an energy source include the inevitable exhaustion of
the world’s fossil oil resources, a possible interruption in oil supply caused by political
interference, the superior net performance of biofuel ethanol in comparison to gasoline, and
a significant reduction in pollution levels. It is to be expected that the demand for
inexpensive, renewable substrates and cost-effective ethanol production processes will
become increasingly urgent.
Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and
waste material) is the only foreseeable sustainable source of fuel ethanol because it is
relatively low in cost and in plentiful supply. The principal impediment to more widespread
utilisation of this important resource is the general absence of low cost technology for
overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to
fermentable sugars from ethanol can be produced. A promising strategy for dealing with this
obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use
in an integrated process, known as direct microbial conversion (DMC) or consolidated
bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF
(separate hydrolysis and fermentation) and SSF (simultaneous saccharification and
fermentation, in which enzymes from external sources are used) in that the production of
polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the
resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting
yeast strain.
The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be
developed that possess the required combination of substrate utilisation and product
formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides
such as starch for the production of high ethanol yields have not been described to date.
However, significant progress towards the development of such amylolytic strains has been
made over the past decade.
With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast
strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes
in S. cerevisiae. This study forms part of a large research programme aimed at improving
these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces
kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1)
and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p
functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In
addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages.
The purpose of this study was two-fold. The first goal was to characterise the molecular
structure of Lka1p and Lka2p in order to better understand the structure-function
relationships and role of specific amino acids in protein function with the aim of improving
their substrate specificity in raw starch hydrolysis. The second aim was to determine the
effect of yeast cell flocculence on the efficiency of starch fermentation, the possible
development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell
biocatalysts’, and the production of high yields of ethanol from raw starch.
In order to understand the structure-function relationships in Lka1p and Lka2p, standard
computational and bioinformatics techniques were used to analyse the primary structure. On
the basis of the primary structure and the prediction of the secondary structure, an N-terminal
region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of
raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p
share a similar catalytic TIM barrel, consisting of four highly conserved regions previously
observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the
catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state
stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p.
Mutational analysis of Q414 performed in the current study provides a basis for understanding
the various properties of Lka1p in relation to the structural differences observed in this
molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties
provides us with the potential to expand the substrate specificity properties of this α-amylase
towards more effective processing of its starch and related substrates.
In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was
used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that
the flocculent cells exhibited physicochemical properties that led to a better interaction with
the starch matrix. This, in turn, led to a decrease in the time interval for interaction between
the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells.
The use of flocculation serves as a promising strategy to best exploit the expression of LKA1
in S. cerevisiae for raw starch hydrolysis.
This thesis describes the approaches taken to investigate the molecular features involved
in the function of the L. kononenkoae α-amylases, and to improve their properties for the
efficient hydrolysis of raw starch. This study contributes to the development of amylolytic
S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel
ethanol from inexpensive starch-rich materials.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1102 |
| Date | 03 1900 |
| Creators | Ramachandran, Nivetha |
| Contributors | Cordero Ortero, Ricardo R., Pretrius, Isak S., University of Stellenbosch. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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