Development of improved α-amylases

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

Amylolytic enzymes from the yeast Lipomyces kononenkoae

The Lipomyces kononenkoae ?-amylases LKA1 and LKA2 belong to the glycoside hydrolase family 13 and exhibit specificity towards ∝-1,4 and ∝-1,6 linkages in starch and related substrates. LKA1 exhibits specificity towards ∝-1,4 and ∝-1,6 linkages and large amounts of reducing sugars are liberated from highly branched amylopectin and glycogen and linear amylose. LKA2, on the other hand, shows high reactivity towards lintner starch, dextrin and amylase, although only small amounts of reducing sugars are liberated from branched substrates, such as amylopectin and glycogen. These enzymes share the four conserved segments of the catalytic domain found in other members of the family, but have some major variant amino acids within these segments. In addition, LKA1 consists of an N-terminal starch-binding domain (SBD). This is the only ∝-amylase known to possess this N-terminal domain and it exhibits homology to the N-terminal SBD of Rhizopus oryzae glucoamylase. It shares no homology with the C-terminal starch-binding domains present in the cyclodextrin glucanotransferases, glucoamylases or ∝-amylases. The evolutionary tree based on the sequence alignment of SBDs reveals that the N-terminal SBDs are separated from the C-terminal SBDs.8 page(s

The Effect of flocculation on the efficiency of raw-starch fermentation by Saccharomyces cerevisiae producing the Lipomyces kononenkoae LKA1-encoded ∝-amylase

A major limitation of most industrially importantSaccharomyces yeast strains are their inability to efficiently convert starch-rich substrates into commercially important commodities, such as bioethanol, low carbohydrate beer and grain whiskey. In an attempt to overcome this impediment, we have previously expressed inSaccharomyces cerevisiae theLKA1 α-amylase-encoding gene from an efficient raw-starch degrading yeast,Lipomyces kononenkoae. Although the engineeredS. cerevisiae strain was capable of utilising starch, the growth rate was much slower than in glucose-containing media and the ethanol yield in batch fermentations was nowhere near the levels required for an economically, viable bioconversion process. The purpose of the present study was to further improve the fermentation performance of the engineered yeast by expressing theLKA1 gene in a flocculent and non-flocculent genetic background. Despite producing similar levels of α-amylase activities in the extracellular culture media, the flocculentS. cerevisiae transformants degraded starch at an earlier hydrolytic window than the non-flocculent transformants. In small-scale batch fermentations, the non-flocculent strain consumed 76% of the starch supplied in the culture medium and produced 4.61 g l⁻¹ of ethanol after 90 h, while the flocculent strain utilised 82% of the starch and produced 5.1 g l⁻¹ of ethanol after 90 h. Flow-cell system and atomic force microscopy revealed that the ‘tighter’ interaction between the flocculent cells and the starch granules might contribute to the better performance of the flocculent transformant.10 page(s

Pediatric Oncology Clinical Trial Participation Where the Geography is Vast: Development of a clinical research system for tertiary and satellite centers in Ontario, Canada.

Opportunities for participation in clinical trials are a core component of the care of children with cancer. In Ontario, many pediatric patients live long distances from their cancer center. This paper describes the work that was done in order to allow patients participating in Children\u27s Oncology Group trials to receive care, including research protocol related care, jointly between the tertiary pediatric cancer center and the closer-to-home satellite center. The system is a pragmatic risk-based model, supporting excellence in care while ensuring good conduct of the research in compliance with applicable regulations and guidelines, including ethics oversight

­­Eleven tips for operational researchers working with health programmes: our experience based on implementing differentiated tuberculosis care in south India

Due to the workload and lack of a critical mass of trained operational researchers within their ranks, health systems and programmes may not be able to dedicate sufficient time to conducting operational research (OR). Hence, they may need the technical support of operational researchers from research/academic organisations. Additionally, there is a knowledge gap regarding implementing differentiated tuberculosis (TB) care in programme settings. In this ‘how we did it’ paper, we share our experience of implementing a differentiated TB care model along with an inbuilt OR component in Tamil Nadu, a southern state in India. This was a health system initiative through a collaboration of the State TB cell with the Indian Council of Medical Research institutes and the World Health Organisation country office in India. The learnings are in the form of eleven tips: four broad principles (OR on priority areas and make it a health system initiative, implement simple and holistic ideas, embed OR within routine programme settings, aim for long-term engagement), four related to strategic planning (big team of investigators, joint leadership, decentralised decision-making, working in advance) and three about implementation planning (conducting pilots, smart use of e-tools and operational research publications at frequent intervals). These may act as a guide for other Indian states, high TB burden countries that want to implement differentiated care, and for operational researchers in providing technical assistance for strengthening implementation and conducting OR in health systems and programmes (TB or other health programmes). Following these tips may increase the chances of i) an enriching engagement, ii) policy/practice change, and iii) sustainable implementation