Journal of Research in Music Education-1978-Chalmers-90-6.pdf
6. Biotechnology - Chalmers
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79 6. Biotechnology
Transcript of 6. Biotechnology - Chalmers
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6. Biotechnology
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Biotechnology
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Metabolic Engineering of Saccharomyces cerevisiae for Isoprenoid Production
Stefan Tippmann1, Sakda Khoomrung1, Verena Siewers1 and Jens Nielsen1
1 Systems & Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
Isoprenoids are a large group of secondary metabolites, which are ubiquitous in the
plant kingdom and have diverse applications in the food, cosmetic and
pharmaceutical industry. In addition, several isoprenoids have been identified as
substitutes for conventional diesel and jet fuels. Since plant extraction as well as
chemical synthesis suffer from several drawbacks and fail to meet industrial
scale, there is a strong demand for an efficient and sustainable production
process. Therefore, metabolic engineering efforts have strongly focused on
establishing cell factories to enable production of these compounds during recent
years.
This project is dedicated to isoprenoids, which can be used as biofuel precursors and
attempts to establish a yeast cell factory for their production. For this purpose,
Saccharomyces cerevisiae was chosen as a host organism, whereas the main focus is
set on sesquiterpenes such as farnesene, which can be used as diesel alternative in its
hydrogenated form farnesane. Different aspects are being addressed in order to
enable for efficient production of farnesene. In the first part, an existing
platform optimized for sesquiterpene production was recently used for the
integration of farnesene synthase genes from different plant sources to enable the
one-step conversion from farnesyl pyrophosphate to farnesene. As a result, final
titers of 170 mg/L were attained in a comparative evaluation in fed-batch
cultivations with exponential feeding. Enhanced synthesis, however, will not only
involve heterologous expression of these enzymes, but it will also include further
engineering of the endogenous mevalonate pathway as well as the integration of
different ’omics’ analysis to support the cycle of metabolic engineering.
Biotechnology
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In situ conversion of phenolic compounds as a tool to phenolic tolerance development by Saccharomyces cerevisiae
Peter Temitope Adeboye, Maurizio Bettiga and Lisbeth Olsson
Industrial Biotechnology Division, Biology and Biological Department, Chalmers University of Technology, SE-41296, Gothenburg, Sweden
Email: [email protected]
Depolymerization of lignin during pretreatment of lignocellulosic materials
usually results in formation of several phenolic compounds. Phenolics are
inhibitory to the growth and function of cells as biocatalysts in the production of
second generation biofuels and chemicals from pretreated lignocellulosic
biomass. Our research is focused on elucidating the metabolic pathways allowing
Saccharomyces cerevisiae to metabolize phenolic compounds. We aim at
understanding the conversion mechanisms of phenolic compounds in S. cerevisiae
and harness them for metabolic engineering towards phenolic tolerant S.
cerevisiae.
We have investigated the inhibitory action of thirteen phenolic compounds against
S. cerevisiae. Our results showed that phenolic compounds have varied inhibition
against S. cerevisiae and the cell response may be dependent on the structure of
the compound involved. Under aerobic batch cultivation conditions, we have also
studied the conversion of phenolic compounds by S. cerevisiae using coniferyl
aldehyde, ferulic acid and p-coumaric acid as phenolic model compounds. We
compiled a list of conversion products of the three compounds under investigation
and we proposed a possible conversion pathway.
With our contribution, we present a proposed conversion pathway through which
S. cerevisiae converts and detoxifies coniferyl aldehyde, ferulic acid and p-
coumaric acid under aerobic cultivation condition.
Biotechnology
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The action of gluceronoyl esterases on Lignin-carbohydrate (LC) ester bonds could assist in a more efficient and native wood
disassembly
Jenny Arnling Bååtha, b, Sylvia Klaubaufa, b and Lisbeth Olssona, b
a Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
bWallenberg Wood Science Center, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
Email: [email protected]
The Swedish pulp and paper industry is facing challenges with a decreased
demand on produced pulp as a result of changes in consumers pattern. Together
with the need for renewable resources to replace oil-based products, the
development of new wood-based materials is of great interest [1]. In order to
produce new materials from wood, the main components (cellulose, hemicellulose
and lignin) have to be extracted in their native and polymeric form in suitable
processes. In these processes, enzyme treatment that targets specific linkages,
could serve as important steps. The chemical linkage pattern proposed to connect
lignin with cellulose and hemicellulose, the so-called lignin-carbohydrate
complexes (LCCs), is an obstacle for an intact and efficient extraction of wood
polymers. However, breakdown of model substrates mimicking LC ester bonds
have successfully been studied with a recently discovered enzyme family,
Glucuronoyl esterases (GEs). These enzymes are proposed to target the ester bond
between the lignin alcohol and the glucuronic acid of carbohydrates in wood [2].
The aim of this work is to study the action of expressed and purified GEs on native
LCCs from spruce, birch and Japanese beech. The extracted LCC fractions will
be treated with GEs and the degradation pattern will be followed by NMR
analysis. The purpose of the study is to obtain more information about the LCC
structure in wood as well as investigating the capacity of GEs as a possible
treatment step in biorefinery processes.
References.
1. N. Westerberg, H. Sunner, M. Helander, G. Henriksson, M. Lawoko, A. Rasmuson. Bioresources. 2012. 7(4): 4501-4516. 2. C. Katsimpouras, A. Bénarouche, D.Navarro, M. Karpusas, M. Dimarogona, J. Berrin, P. Christakopoulous, E. Topakas. Appl. Microbiol. Biotechnol. 2014. 98: 5507-5516.
Biotechnology
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Moderate Overexpression of SEC16 Improves α-Amylase Secretion in Saccharomyes cerevisiae
Jichen Baoa, Mingtao Huanga, Dina Petranovica and Jens Nielsena,b
aNovo Nordisk Foundation Center for Biosustainability, Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden bNovo Nordisk Foundation Center for Biosustainability, Technical University of
Denmark, Hørsholm, Denmark [email protected]
There is a large and increasing demand of recombinant proteins, not only for
pharmaceuticals but also in the field of industrial enzymes (Huang, Bao et al.
2014). Recombinant proteins can be produced by a range of different hosts, ,
including mammalian cells, insect cells, bacteria, yeasts and fungi (Hou, Tyo et
al. 2012). Each of these have their own advantages and disadvantages, and none
would naturally make a suitable general platform for producing of a wide range
of recombinant proteins, with satisfactory yield, titer and productivity. Therefore,
host optimization is required for the development of efficient recombinant protein
producers.
Yeast Saccharomyces cerevisiae is one of preferred model microbial and eukaryal
systems, because of its robustness, well-studied genetics and physiology,
developed molecular tools and large free databases. The limitations sometimes
include translation and sometimes the folding and secretory capacity, which is
more challenging to address. In this study, we focused on the secretory pathway
and improved the secretion of a model enzyme α-amylase by overexpressing
SEC16, which is a COPII vesicle coat protein required for ER to Golgi vesicle
budding and transport . When SEC16 was overexpressed from a low copy number
plasmid with the strong constitutive promoter TEF, there was ~ 60% improvement
in the final titer of α-amylase. Interestingly, when SEC16 was overexpressed from
a high copy number plasmid with the strong TEF promoter, there was no
improvement. The result indicates that the positive effect of overexpressing
SEC16 on α-amylase secretion is dosage dependent, and suggests that COPII
vesicle formation is not a very limiting step in our system, so we will also focus
on other proteins that might be more limiting in the secretion pathway.
References.
Hou, J., et al. (2012). "Metabolic engineering of recombinant protein secretion by
Saccharomyces cerevisiae." FEMS Yeast Res 12(5): 491-510.
Huang, M., et al. (2014). "Biopharmaceutical protein production by
Saccharomyces cerevisiae: current state and future prospects." Pharm. Bioprocess
2(2): 167-182.
Biotechnology
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Immobilization in MPS and characterization of a FAE for hydrolysis and transesterification reactions
Cyrielle Bonzom, Laura Schild and Lisbeth Olsson
Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, Göteborg SE
41296, SWEDEN. Phone: +46317723843
Email: [email protected]
Mesoporous silica materials (MPS) are an interesting choice as support to
immobilize enzymes because MPS offer unique properties such as high enzyme
loading and tunable pore size. They also provide the enzyme with a sheltered
environment therefore reducing the risks of denaturation in industrial
applications.
Immobilization parameters such as pH, buffer and pore size of the material were
investigated. Among them, the chemical composition of the buffer as well as its
pH proved to be critical resulting in enzyme loadings varying from nearly zero up
to 0.025 mgenzyme.mgMPS-1.
Selectivity of the enzyme, a feruloyl esterase (FAE), was investigated by
quantifying the molar ratio between the transesterification and hydrolysis
products, namely butyl ferulate (BFA) and ferulic acid (FA). The reaction of
interest was transesterification therefore hydrolysis was an unwanted side-
reaction. The immobilization pH and the water content of the reaction were the
most influent parameters inducing variation up to 4-fold of the BFA/FA molar
ratio.
Optimal reaction conditions and kinetic parameters of the free and immobilized
enzyme were determined for both hydrolysis and transesterification to determine
in which conditions transesterification is prevailing. While optimal pHs were
similar for all studied, temperature optimums varied from 25 to 50°C.
Interestingly the Km of the FAE was not affected upon immobilization, but the kcat
was decreased 10-fold resulting in a lower catalytic efficiency. Km was 100-fold
higher for transesterification than for hydrolysis whereas kcat was 100-fold lower;
this resulted in a drastic reduction of the catalytic efficiency of the FAE.
Stability of the enzyme was evaluated using the hydrolysis reaction. No
significant improvement could be observed for the immobilized enzyme.
Reusability of the immobilized biocatalyst was determined during 10-cycles of
48h. A decrease in activity was observed during the course of the experiment. In
addition a decrease in the BFA/FA molar ratio indicating a shift in enzyme
specificity happened.
Biotechnology
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Smart drug delivery materials
Marina Craiga,b,c and Krister Holmbergb,c
aMölnlycke Health Care, Box 130 80, 402 52 Göteborg bChalmers University of Technology, 412 96 Göteborg
cSuMo Biomaterials, Chalmers University of Technology, 412 96 Göteborg Email: [email protected]
The development of soft biomaterial assembly has in recent years taken a great leap forward. In the current study we have investigated the use soft
materials in drug release triggered by infection. The skin flora, i.e. the human
microbiome, is a positive and beneficial colonization of bacteria on skin and
is necessary for our survival.[1] However, compromised or broken skin can
create a passage for species that are pathogenic in deeper tissue or in blood. An example is patients with lifestyle diseases such as diabetes or venous
insufficiency that often acquire chronic wounds. Such wounds are hard to
heal due to the underlying disease and poor blood circulation, which in turn
contributes to a favourable environment for bacteria and thus infection. By
treating part of the infection locally issues with bacterial resistance and
overexposure of systemic drugs can be targeted. Additionally, the bacteria
continuously exude substrate specific proteases to degrade host tissue and hence provide nutrition for the pathogens. The amount of proteases is
dependent on the size of the bacterial colonies, with a clinically infected
wound carrying a rather high protease concentration. With this information
at hand, two nanofilms were assembled (layer-by-layer) as shells on
microcapsules (template assisted assembly) for infection triggered
degradation when exposed to virulent strains of either Staphylococcus
aureus[2-4] or Pseudomonas aeruginosa.[5] An antimicrobial drug was then
loaded into the film and core after the sacrificial template was dissolved. Antimicrobial tests on bacteria (and other tests) showed great promise for a
release platform responding to a specific bacterium as well as the
concentration of the protease, while the film remained intact if no infection
was present (no drug release).
References.
[1] K. Todar. (2012) The online textbook of bacteriology. www.textbookofbacteriology.net [2] M. Craig, R.Bordes, K. Holmberg. (2012) Soft Matter 8 (17) 4788. [3] M. Craig, E. Schuster, K. Holmberg. (2014) Accepted in Materials Today Proceedings [4] M. Craig, M. Amiri, K. Holmberg. (2015) Coll. Surf. B 127, 200. [5] M. Craig, A. Altskär, L. Nordstierna, K. Holmberg. Submitted to Advanced Functional Materials
Biotechnology
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Life under stress: Using novel tools to elucidate yeast stress responses
Eugene Fletchera, Petri-Jaan Lahtveea and Jens Nielsena
a Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, Gothenburg, Sweden
Email: [email protected], [email protected]
Engineered yeasts are currently being used as a more preferred cell factory for the
industrial production of new bio-based products from feedstocks. Utilization of
various substrates, high production rates and robustness towards various stress
factors are the main properties expected from an efficient producer strain.
Therefore, to circumvent the latter limitation, we have established two main
streamlines to be applied independently or concomitantly: 1) adaptive laboratory
evolution (black-box model) and 2) systems biology of cellular regulation (white-
box model). While in the first model an a priori knowledge is not required to
enhance cell’s robustness, the second model drives the cell’s improvement via
heuristic-based methods which require a high-level understanding of cell
metabolism and regulation. Adaptive laboratory evolution (ALE) was used to
select strains of Saccharomyces cerevisiae (bakers’ yeast) with improved growth
at pH 2.8 and by re-sequencing the whole genome of the evolved strains,
important genes involved in acid stress have been identified. Using an alternative
approach, response towards three industrially relevant stresses, namely ethanol,
salt and temperature, were studied in detail on a molecular level. By integrating
quantitative transcriptome and proteome analysis with metabolic modelling, we
detected an increased energy maintenance resulting from increased membrane
permeability, intracellular oxidative stress or increased protein turnover as the
main reason for decreased growth efficiency under the studied conditions.
Ultimately, we hope to understand the molecular mechanisms behind stress
regulation in yeast and by employing the reverse engineering tools, more efficient
cell factories for the production of bio-chemicals can be developed.
Biotechnology
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Characterization of a novel non-GMO yeast for future bioethanol production
Cecilia Geijer, David Moreno, Elia Tomas Pejo and Lisbeth Olsson.
1Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
2Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), Spain
The urgency to reduce carbon emissions and lower our dependence on oil makes
it necessary to strive towards a more sustainable, bio-based economy where
energy, chemicals, materials and food are produced from renewable resources.
Lignocellulose derived from plant biomass constitutes a great source of raw
material for such a future bio-based economy because it is widely available,
relatively inexpensive and do not compete with food and feed production. The
yeast Saccharomyces cerevisiae display excellent glucose fermenting skills, but
metabolic engineering is needed to allow consumption and fermentation of xylose
(the second to glucose most prevalent sugar in lignocellulose). We have isolated
a non-genetically modified (non-GMO) yeast species (here called C5-yeast) that
has the natural ability to efficiently produce ethanol from glucose and xylose. The
aim of the project is to further characterize the growth and fermentation capacities
of this novel microorganism to elucidate its potential for lignocellulosic
bioethanol production. We can show that besides glucose and xylose, the C5-yeast
can consume a wide range of sugars including the pentose arabinose and the
disaccharide cellobiose; both present in lignocellulosic hydrolysates. De novo
genome assembly of the C5 yeast will hopefully provide further insights into the
physiology of the yeast.
Biotechnology
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FUNGAL glucuronoyl and feruloyl esterases for wood processing and phenolic acid ester/sugar ester synthesis
Silvia Hüttnera, *, Sylvia Klaubaufb, Hampus Sunnerb, Cyrielle Bonzoma, Peter Jüttenc, and Lisbeth Olssona, b
a Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
b Wallenberg Wood Science Centre, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
c Taros Chemicals GmbH & Co. KG, 44227 Dortmund
Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases (GEs,
E.C. 3.1.1.-, CAZy family CE15) are involved in the degradation of plant biomass by
hydrolysing ester linkages in plant cell walls, and thus have potential use in biofuel production
from lignocellulosic materials and in biorefinery applications with the aim of developing new
wood-based compounds [1, 2]. GEs and FAEs are present in the genomes of a wide range of
fungi and bacteria.
Under conditions of low water content, these enzymes can also carry out (trans)esterification
reactions, making them promising biocatalysts for the modification of compounds with
applications in the food, cosmetic and pharmaceutical industry. Compared to the chemical
process, enzymatic synthesis can be carried out under lower process temperatures (50-60°C)
and results in fewer side products, thus reducing the environmental impact.
We characterised new FAE and GE enzymes from mesophilic, thermophilic and cold-tolerant
filamentous fungi produced in Pichia pastoris. The enzymes were characterised for both their
hydrolytic abilities on various model substrates (methyl ferulate, pNP-ferulate) - for potential
applications in deconstruction of lignocellulosic materials and extraction of valuable
compounds - as well as for their biosynthetic capacities. We tested and optimised the FAEs’
transesterification capabilities on ferulate esters in a 1-butanol-buffer system, with the aim of
using the most promising candidates for the production of antioxidant compounds with
improved hydrophobic or hydrophilic properties, such as prenyl ferulate, prenyl caffeate,
glyceryl ferulate and 5-O-(trans-feruloyl)-arabinofuranose.
Acknowledgments: Esterase research at Chalmers is supported by the EU Framework 7 project
OPTIBIOCAT, the Knut and Alice Wallenberg Foundation (Wallenberg Wood Science Center)
and the Swedish Research Council (Linnaeus Centre for Bio-inspired Supramolecular Function
and Design, SUPRA).
____
[1] Topakas, E., Vafiadi, C., and Christakopoulos, P. (2007). Microbial production, characterization and
applications of feruloyl esterases. Process Biochemistry, 42(4), 497–509. doi:10.1016/j.procbio.2007.01.007
[2] Spániková, S., and Biely, P. (2006). Glucuronoyl esterase - Novel carbohydrate esterase produced by
Schizophyllum commune. FEBS Letters, 580(19), 4597–601. doi:10.1016/j.febslet.2006.07.033
Biotechnology
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Extraction and hydrolysis of algal laminarin for ethanol fermentation
Joakim Olsson, George Anasontzis, Eva Albers, Jenny Veide Vilg
Chalmers University of Technology, Dept. Biology and Biological Engineering/Industrial Biotechnology, Gothenburg, Sweden
Email: [email protected]
Seaweeds contain high levels of carbohydrates with potential for further
bioprocessing into valuable commodities. In the SEAFARM project, brown
seaweed cultivated on the Swedish West coast will be subjected to biorefining
with the aim to maximise its value as a raw material for functional foods,
nutraceuticals, biomaterials, feed and bioenergy. The brown seaweeds Saccharina
latissima and Laminarina digitata contain up to around 20% (DW) of the ß-1,3-
glucan laminarin, depending on season. To convert the laminarin into ethanol by
fermentation, it needs to be extracted and then hydrolysed to glucose units. In
SEAFARM, we aim to develop extraction methods for laminarin that are
compatible with other processes within the project. We are also exploring bacteria
for novel laminarin-degrading enzymes, as an alternative to energy-demanding
acid hydrolysis. Preliminary results show functional enzymatic degradation in a
simultaneous saccharification and ethanol fermentation by yeast, with ethanol
yields comparable with those of fermentations following acid hydrolysis.
Biotechnology
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Transcription factors and their role in gene regulatory networks
David Jullesson, Guodong Liu and Jens Nielsen
Chalmers University of Technology, Department of Biology and Biological Engineering, Division of Systems and Synthetic
Biology
Email: [email protected]
Understanding the underlying role of cellular functions is needed to understand
how the cell is regulated. Examining the transcription factor (TF) regulation and
how they regulate gene expression in different growth conditions and to which
motifs and promoter regions the TFs bind to can lead to understanding the cell
regulation. To investigate this regulation a system for tagging the transcription
factor of interest were built to further extract the TF of interest. The model
organism is the yeast Saccharomyces cerevisiae which is widely used in industrial
processes for production of ethanol and also other high value chemicals. For this
reason the aim of this project is to create more robust and better predictable cell
factories. The modified yeast cells are cultivated in four different chemostat
conditions which all are interesting in an industrial perspective. For the best
performance of signaling motifs the technique ChIP-exo (Rhee and Pugh, 2011)
(Chromatin ImmunoPrecipitation sequencing using exonuclease) is used. ChIP-
exo is the newest state-of-the-art method for sequencing motifs. The method
involves a tagged TF, DNA shearing, digestion using exonuclease and
sequencing. The data will be analyzed and a signaling and gene regulation model
will be built or incorporated into already existing models. Figure.
This figure represent 4 different growth conditions with biological duplicates for each conditions. The
binding sites for the targeted transcriptionfactor (INO2) can be seen as peaks. The data shows good
duplication between the experiments and a difference in binding between the growth conditions can be
seen.
References.
Rhee, Ho S., Pugh, B. F. Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution. Cell. 2011;147:1408-1419.
Biotechnology
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Structural changes of cellulosic materials during enzymatic hydrolysis studied by nonlinear microscopy
Juris Kiskisa, Ausra Peciulytea, Lisbeth Olssona,b and Annika Enejdera
a Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
b Wallenberg Wood Science Center, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
E-mail: [email protected]
Liberation of fermentable soluble sugars from cellulosic biomass during the
course of enzymatic hydrolysis is the major obstacle to large-scale
implementation of biorefineries due to high cost of enzymes. Enzymatic
hydrolysis of cellulosic biomass is often incomplete and, therefore, it is of great
importance to understand the limitations of the process. Among the limitations of
enzymatic hydrolysis, structural properties of cellulose have an effect on
enzymatic hydrolysis efficiency. Currently, there is a lack of direct methods for
visualization and quantification of spatial polymer distribution in cellulosic
biomass and monitoring of interactions between cellulose degrading enzymes and
the substrate. In the current work, we used nonlinear microscopy to visualize
cellulosic materials during the process of enzymatic hydrolysis. The overall aim
was to contribute to understanding of relation between enzymatic hydrolysis and
structural properties of cellulosic materials at the micro scale.
Enzymatic hydrolysis during the course of several hours was studied on three
cellulosic materials: cellulose fiber, nano-crystalline cellulose (NCC) and
commercial micro-crystalline cellulose (Avicel) particles. These materials were
imaged with nonlinear optical microscope, employing multi-photon excitation
fluorescence (MPEF), second-harmonic generation (SHG) and coherent anti-
Stokes Raman scattering (CARS) modalities. Hydrolysis of cellulose fiber and
NCC particles was more prominent compared to the particles of Avicel. Changes
of the shape of cellulose fiber and NCC particles were observed during hydrolysis.
We have investigated possible relation of the change in shape to the structure of
the cellulosic materials prior to enzymatic hydrolysis and underlying structural
changes during enzymatic hydrolysis.
Biotechnology
93
Metabolic engineering of Bacillus subtilis for the production of 3-hydroxyproponic acid
”Aida Kalantari 1a,b,c, Anne Goelzer 2c and Ivan Mijakovic 3a
a Chalmers University of Technology, Sys Bio, Gothenburg, Sweden b Chaire Agro-Biotechnologies Industrielles (ABI) AgroParisTech, Reims, France c Unité de Mathématique, Informatique et Génome (MIG) Jouy-en-Josas, France
Email: [email protected]
Over the past few years a major interest is focused on the substitution of
petrochemical products by processing bio-products from renewable raw
materials. In this context, our project focuses on the production of 3-
Hydroxypropionic acid (3-HP) from glycerol. 3-HP is one of the top-valued
platform chemicals used for the production of many industrially important
chemicals such as acrylic acid, and glycerol is a relatively cheap byproduct of
biodiesel industry. 3-HP can be produced by several microorganisms which
possess glycerol dehydratase (dhaB) and aldehyde dehydrogenase (aldH) genes,
however, bio-production of 3-HP has never reached a commercially exploitable
level. In this project, we propose to explore the possibility of synthesizing 3-HP
for the first time in Bacillus subtilis by developing a novel and more efficient
pathway. Our genetically engineered B. subtilis will contain dhaB and aldH genes
from other organisms like Lactobacillus reuteri, Klebsiella pneumonia and
Clostridium butyricum. Metabolic network will be optimized in silico to guide our
metabolic engineering strategy in vivo.
References.
Recent advances in biological production of 3-hydroxypropionic acid (Kumar et al) 2013) 1. Bacterial growth rate reflects a bottleneck in resource allocation (Goelzer et al 2011) 2.
Biotechnology
94
Metabolic engineering and search for novel enzymes for bio-based production of adipic acid
Emma Karlssona, Luigi D’Avinoa, Lisbeth Olssona,Valeria Mapellia
aIndustrial Biotechnology, Chalmers University of Engineering, Kemigården 4, 412 96 Gothenburg, Sweden
Email: [email protected]
Adipic acid is a six carbon long dicarboxylic acid, considered to be the most
important synthetic dicarboxylic acid annually produced, according to the
International Energy Agency (IEA). The global production of adipic acid had in
2010 a volume of 2.8 million tonnes, for a total market price of 4.9 billion USD.
The current production of adipic acid relies on non-renewable fossil raw
materials, leading to emission of the greenhouse gases CO2 and N2O. In addition,
the production starts from benzene, whose use has several health related negative
implications. This project aims to create a greener process for production of adipic
acid developing a fermentation-based process using Swedish domestic renewable
raw materials, such as forest residues and algae. These materials will be used to
establish a biorefinery, wherein the fermentation process for the biosynthesis of
adipic acid will represent the core process. The metabolic pathway of our choice
for production of adipic acid is presented: lysine is converted into adipic acid in
four enzymatic steps, without directly competing with the central carbon
metabolism and the pathway is balanced regarding cofactors. For the three first
enzymatic steps the reactions are known but there are today no known enzymes
performing the desired reactions. In this project the focus is therefore to identify
suitable enzymes for these three steps. The conversion of glucose to lysine, with
high yields, is already established in Corynebacterium glutamicum and hence this
bacterium is an attractive target for introduction of this pathway. However, as a
proof-of concept for bio-based production of adipic acid the generation of
genetically modified strains of the yeast Saccharomyces cerevisiae, harbouring
heterologous enzymatic activities allowing the conversion of lysine into adipic
acid is our first choice. Here we present our metabolic engineering strategy along
with preliminary results from expression of heterologous genes of choice and
effect of adipic acid on S. cerevisiae physiology.
Biotechnology
95
New glucuronoyl esterases for wood processing
Sylvia Klaubaufa, Silvia Hüttnerb, Hampus Sunnera and Lisbeth Olssona,b
aWallenberg Wood Science Centre, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
bDepartment of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
Email: [email protected]
The development of new wood-based materials is of great interest to the forest
industry. Wood tissue is composed of a complex biopolymer mixture containing
cellulose, hemicellulose and lignin. Covalent bonds between lignin and different
polysaccharides form closely associated structures known as lignin-carbohydrate
complexes (LCCs). As a result, the successful extraction and separation of wood
polymers poses a major challenge for materials biorefinery concepts. Enzymes
that target lignin-carbohydrate (LC) bonds are especially useful for biorefinery
applications as they can facilitate the isolation of individual wood components in
combination with mild chemical treatments. The main LCCs present in wood are
believed to be esters, benzyl ethers and phenyl glycosides [1,2]. Glucuronoyl
esterases (GEs) have been proposed to degrade ester bonds between glucuronic
acids in xylans and lignin alcohols. GEs belong to the carbohydrate esterase (CE)
15 family and are present in the genomes of a wide range of fungi and bacteria.
The aims of our study were to characterize new GE enzymes, to investigate their
capacity in disconnecting hemicellulose from lignin and to apply them in the
extraction process. Selected candidate genes encoding novel GEs from a diverse
range of filamentous fungi were produced and tested on model substrates as well
as LCC fractions and their applicability in wood processing is investigated.
References.
1. Balakshin MY, et at. (2007) MWL fraction with a high concentration of lignin-carbohydrate linkages:
Isolation and 2D NMR spectroscopic analysis. Holzforschung 61:1–7.
2. Watanabe T (1995) Important properties of lignin-carbohydrates complexes (LCCs) in
environmentally safe paper making. Trends in Glycoscience and Glycotechnology 7:57–68.
Biotechnology
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Membrane engineering for reduced acetic acid stress: insights from Zygosaccharomyces bailii
Lina Lindahla, Aline X S Santosb, Samuel Genhedenc, Leif A Erikssond, Howard Riezmanb, Lisbeth Olssona, Maurizio Bettigaa
aDepartment of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
bDepartment of Biochemistry, University of Geneva, Geneva, Switzerland cSchool of Chemistry, University of Southampton, Southampton, UK
dDepartment of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden
Email: [email protected]
The high concentration of acetic acid released during pretreatment of
lignocellulose raw material is a major obstacle to the microbial production of bio-
based products. Acetic acid enters the cell mainly by passive diffusion across the
plasma membrane and inhibits yeast by mechanisms such as reduction of
intracellular pH, accumulation of the acetate anion, and by signaling effects
triggering cell death.
Through extensive characterization of the acetic acid tolerant yeast
Zygosaccharomyces bailii, we have identified the cell membrane as a target for
strain engineering with potential to increase acetic acid tolerance in
Saccharomyces cerevisiae.
We propose membrane permeability as a key component for Z. bailii’s acetic acid
tolerance. We have previously shown that Z. bailii has a unique ability to remodel
its plasma membrane upon acetic acid stress, to strongly increase its fraction of
complex sphingolipids, at the expense of a drastic reduction of
glycerophospholipids [1].
Here we further demonstrate the involvement of complex sphingolipids in acetic
acid tolerance by decreasing sphingolipid synthesis using the drug myriocin, and
characterize the acetic acid tolerance in terms of growth and intracellular pH.
Furthermore we show the impact of complex sphingolipids on membrane physical
properties using in silico membrane simulations. Ongoing membrane engineering
of S. cerevisiae can potentially give additional strength to our findings.
References
[1] Lindberg et al. (2013), Lipidomic Profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii Reveals Critical Changes in Lipid Composition in Response to Acetic Acid Stress, PLoS One 8: e73936.
Biotechnology
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Characterization of metabolism and robustness of Lactococcus lactis under different fermentation conditions for production of
starter cultures with improved robustness
Bettina Lorantfy, Valeria Mapelli, Carl Johan Franzén and Lisbeth Olsson
Division of Industrial Biotechnology, Department of Biology and Biological Engineering - Chalmers University of Technology, Göteborg, Sweden
Email: [email protected]
Lactic acid bacteria have been used for cheese making for thousands of years,
thanks to their capability for milk acidification; for the anaerobic production of
acidic by products on the sugar content of the milk. Nowadays, in the dairy
industry, starter cultures are commercially produced for the cheese making mainly
via anaerobic batch fermentations. The fermentation part of the production is
followed by a chain of downstream operations, where starter cultures are, for
example, freeze-dried. It was recently discovered that lactic acid bacteria are able
to sustain respiratory metabolism when an exogenous source of hemin, an iron
containing heterocyclic molecule, is added to the growth medium under aerobic
conditions. Hemin allows in fact the electron transport chain for respiration to be
completed. Respiration is energetically beneficial for the cells, and under
respiratory conditions, higher biomass yield is produced and a by-product pattern
different than the one obtained under fully fermentative metabolism is
experienced. The aim of this project is to investigate how different culture
conditions; anaerobic, aerobic, and respiratory of lactic acid bacteria influence the
robustness of the cells. Combining batch and continuous cultivations, and
applying different omics-techniques, the main objective of this work is to find
possible robustness markers for the freeze dried starter culture end product.
Preliminary results showed that with the addition of exogenous hemin, cells
switch from fermentative to respiratory metabolism only in the late exponential
phase of growth, following the usual fermentative behaviour. Although presence
of oxygen is an additional stress for lactic acid bacteria, in the presence of hemin
under aerobic conditions cells have surprisingly better fermentation performance
before the switch to respiration, compared to cells growing in the absence of
hemin. Therefore, it is hypothesized that a certain energy threshold must be
achieved for switching to respiration and this threshold is achieved via improved
fermentation. The energy threshold to be achieved could be for example related
to the necessary energy for the intake of the hemin, i.e. its transport to the cell has
not been characterized yet. Better scientific understanding of the switching to
respiration could help in finding answers to changes in the culture performance
between the different cultivation conditions in connection to robustness.
Biotechnology
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Biocompatible Nanocellulose Hydrogels for 3D Bioprinting of Tissue Constructs
Athanasios Mantas, Guillermo Toriz Gonzalez, Daniel Hägg and Paul Gatenholm
Biopolymer Technology, Dept. of Chemistry and Chemical Engineering, Chalmers University of Technology, Göteborg, Sweden
Email: [email protected]
The objective of this research has been to prepare bioink based on bacterial
nanocellulose. Bacterial nanocellulose (BNC) hydrogel is an emerging
biomaterial in tissue engineering applications due to its biocompatibility and
tissue integration. This natural polymer, produced by the aerobic bacterium
Gluconacetobacter xylinus, is composed of highly crystalline and hydrated (99%
water) nanosized cellulose fibrils with high mechanical strength1. Due to its
outstanding properties BNC has been used already commercially as an FDA
approved wound care product. In a recent study BNC was used clinically as a graft
for dural replacement in 62 patients. BNC is synthesized extracellularly as
nanosized fibrils when the bacteria utilize glucose as a source and form an
exopolysaccharide. The cellulose chains are hold together due to the hydrogen
bonds of the hydroxyl groups. Due to its high crystallinity the mechanically strong
3D nanosized network of BNC is difficult to be converted and be used as a bioink
for bioprinting applications. We have developed an efficient homogenization
process which combines chemical, enzymatic and mechanical treatment to
convert 3D network into bioink with shear thinning properties which provide
outstanding results in printability and shape fidelity. Furthermore the bioprinting
of BNC with alginate and fibroblasts produced 3D crosslinkable constructs
(Figure 1). The cell viability after 7 days proved to be high (79%) indicating great
future for BNC as a bioink in tissue engineering applications.
Figure 1. 3D bioprinted ear from bacterial nanocellulose and alginate (left) 3D printed ear from
polylactic acid (PLA) (right).
References.
1. Martínez Ávila H, Feldmann E-M, Pleumeekers MM, Nimeskern L, Kuo W, de Jong WC, et al. Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo. Biomaterials. 2015;44:122-33
Biotechnology
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An integrated microalgal biorefinery concept – combined pigment production and fermentation feedstock generation
Joshua Mayers, Eva Albers
Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Goteborg
Microalgal cultivation is an attractive platform for the manufacture of biobased
products and materials, ranging from bioenergy feedstocks and bulk chemicals to
high-value molecules. In addition, microalgae can be cultivated on non-drinkable
water sources, recover nutrients from waste streams and can fix CO2 from flue-
gas emissions1. This provides an ideal opportunity for integration with other bio-
processes.
High-value pigment and antioxidant production in green microalgae is a
promising due to increased demand across a range of sectors, scarcity of natural
sources, and unsustainable practices in current agricultural system. These
products include the orange pigments, lutein and β-carotene, both with large
markets, with the former having an annual estimated growth of 3.6%2. Microalgae
may subsequently provide a more sustainable, less resource intensive and
economically stable route for producing these compounds.
The residual materials following pigment extraction from algae are rich in
essential amino acids, monosaccharides and vitamins, all of which are potentially
suitable as a nutrient source for yeast fermentation. By integrating these processes,
reduced reliance on costly sources of organic carbon and nitrogen containing
compounds could be achieved. Additional integration of these platforms with
anaerobic digestion and lignocellulosic processes may further enhance this
biorefinery scenario and development development of a circular economy with
regards to essential nutrient recycling (carbon, nitrogen and phosphorus).
This poster will present a study on a possible biorefinery process for microalgal
pigment production, likely input requirements and how the proposed integration
with other industrial bioprocesses can offset them. References.
1) Wijffels, et al., 2010. Microalgae for the production of bulk chemicals and biofuels.
Biofuel. Bioprod. Bioref. 4: 287-295.
2) Lin, et al., 2014. Lutein production from biomass: marigold flowers versus microalgae.
Bioresour. Technol. 184:421-428.
Biotechnology
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Biosynthetic potential and comparative genomics of the Penicillium genus
Jens Christian Nielsen1* and Jens Nielsen1
1Department of Biology and Biological Engineering, Chalmers University of technology, Gothenburg, Sweden
*Email: [email protected]
Production of pharmaceutically relevant secondary metabolites (SMs)
is a characteristic of Penicillium species, which has formed the
foundation for a lot of research within the genus. The beneficial effects of some of these compounds have been industrially exploited for
decades with the penicillin antibiotics, derived from Penicillium
chrysogenum, being the prime example. With the rise of the genomic era and development of genome mining strategies for SM discovery, it
has been revealed that the array of SMs produced by filamentous fungi
under normal laboratory conditions constitute only a fraction of the genetically encoded diversity [1].
This project aims at uncovering the full biosynthetic potential of SMs
of the Penicillium genus, at a genomic level. Whole genome sequencing of has been conducted for 10 selected Penicillium strains known to
produce a great diversity of SMs [2]. The genomes will be assembled and analyzed with emphasis on SM biosynthetic potential and
compared to already sequenced members of the genus. Focus will be on the evolutionary aspects and horizontal gene transfer events of SM gene
clusters as well as on the phylogeny within the genus.
References. [1] Wiemann and Keller (2014). J Ind Microbiol Biotechno 41:301–313.
[2] Frisvad et al. (2004). Studies in Mycology 49: 201-241.
Biotechnology
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Nonlinear microscopy and CP/MAS 13C-NMR as tools for studying structural changes of cellulosic substrates during enzymatic
hydrolysis
Ausra Peciulytea, Juris Kiskisb, Katarina Karlströmc, Annika Enejderb, Per Tomas Larssonc,d and Lisbeth Olssona
a Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
b Department of Biology and Biological Engineering, The Group of Molecular Microscopy, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
c Innventia AB, SE-114 86 Stockholm, Sweden d Wallenberg Wood Science Center, KTH, SE-100 44 Stockholm, Sweden
e Wallenberg Wood Science Center, Chalmers, SE-100 44 Stockholm, Sweden
Email: [email protected]
Liberation of fermentable sugars from cellulosic biomass during the course of
enzymatic hydrolysis is one of the major obstacles in biorefineries due to high
cost of enzymes. Enzymatic hydrolysis of cellulosic biomass is often incomplete.
Understanding the limitations of the process would aid in improving the process.
Among the limitations of enzymatic hydrolysis, structural properties of cellulose
have a large effect on enzymatic hydrolysis efficiency. The aim of this study was
to increase the understanding of the relation between enzymatic hydrolysis and
structural properties of cellulosic substrates.
In the current work, different cellulosic substrates were imaged and structural
changes of cellulosic substrates were characterized in real-time in micrometer
scale with nonlinear optical microscope, employing multi-photon excitation
fluorescence (MPEF), second-harmonic generation (SHG) and coherent anti-
Stokes Raman scattering (CARS) modalities. Solid-state cross-polarization magic
angle spinning carbon-13 nuclear magnetic resonance (CP/MAS 13C-NMR) was
employed to quantify the spatial polymer distribution, accessible surface area,
crystallinity and porosity in cellulosic substrates in nanometer scale. An array of
cellulosic substrates was used in our study coming from softwood preparations
used in pulp and paper industry and some model substrates.
A strong correlation was found between the average pore size of the starting
material and the enzymatic conversion yield. The degree of crystallinity was
maintained during enzymatic hydrolysis of the cellulosic substrates. A substrate
depended hydrolysis pattern was observed during enzymatic hydrolysis in real-
time. The substrate depended hydrolysis pattern was interpreted in terms of the
underlying structural changes in micrometer and nanometer scale.
Biotechnology
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Modulation of oxidative folding to improve recombinant protein production in the yeast Saccharomyces cerevisiae
Markus M.M. Bisschopsa, Dina Petranovicb Jens Nielsena,b
aSystems and Synthetic Biology, Department of Biology and Biological Engineering & The Novo Nordisk Foundation Center for Biosustainability, Chalmers University of
Technology, Göteborg, Sweden; bThe Novo Nordisk Foundation Center for Biosustainability, Technical University of
Denmark, Hørsholm, Denmark
The yeast Saccharomyces cerevisiae is widely used in biotechnology for the
production of both bulk chemicals, like ethanol and organic acids, and higher-
value compounds such as recombinant proteins, like pro-insulin. Recombinant
protein production in S. cerevisiae induces different stress responses, including
oxidative stress response. This is especially the case when the overall protein
folding rate and the oxidative folding rate, i.e. the formation of di-sulfide bonds,
are not properly balanced (1). In the present study we aim to develop metabolic
engineering strategies to increase recombinant protein production by specifically
adjusting oxidative folding. Two industrially relevant proteins are selected based
on different overall folding rates: insulin-precursor and alpha-amylase. The
oxidative folding of these proteins was modulated by A) altering the expression
levels of several key players in oxidative folding, e.g. the thiol oxidases Ero1 and
Erv2, and B) changing the number of possible disulfide bonds, i.e. the number of
cysteine residues present. The effects of these modulations on ROS levels,
unfolded protein response, oxidative state, protein titers and overall physiology
are monitored under strictly controlled growth conditions.
References
1. Tyo K.E., Z. Liu, D. Petranovic, J. Nielsen (2012) Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress. BMC Biol. 10:16
Biotechnology
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Can we use metabolic modelling to predict enzyme usage in yeast?
Benjamín J. Sáncheza, Cheng Zhanga,b and Jens Nielsena
aDepartment of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
bState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
Email: [email protected]
Genome scale models (GEMs) are a recurrent approach for modelling cellular
metabolism, i.e. the sum of all chemical reactions that occur inside of a cell. These
models allow to make predictions of each reaction’s flux, i.e. the rate at which each
reaction is working1. A current challenge in GEMs is to connect fluxes to the
corresponding enzyme concentrations inside the cell. The connection between
reactions and enzymes can be symbolized by equation 1.
v ≤ [E] ∙1
MW∙ kcat [Eq. 1]
Where v is the reaction’s flux [mmol/gDWh], [E] the enzyme concentration inside
the cell [g/gDW], MW the enzyme’s molecular weight [g/mmol] and kcat the
enzyme’s speed when it is working at saturation, i.e. full capacity [1/h]. Although
the connection between metabolism and enzymes seems straightforward, only a
handful of studies2 so far have tried to merge both concepts into the same modelling
framework, mainly because of (i) the difficulty of obtaining experimental
measurements of all enzymatic concentrations, (ii) the high variation of kcat values in
online databases, (iii) the lack of annotation in GEMs for relating reactions with
enzymes, and (iv) the complexity of some reaction-enzyme relations, such as enzyme
promiscuity (one enzyme can catalyse several reactions) or isozymes (several
enzymes can catalyse the same reaction).
Here we present a method for enhancing metabolic modelling with enzyme usage,
based on the constraint-based approach3, but extended to include enzymes as part of
the reactions, therefore using elemental mass balances for all metabolites and
enzymes inside the cell. Additionally, non–ideal cases such as isozymes and
promiscuous enzymes are taken into account using logical transformations. We show
how the method works on a Saccharomyces cerevisiae (budding yeast) model, and
present some preliminary results such as the prediction of total enzyme usage in
yeast. The method will be used for analysing experiments with all enzyme
concentrations measured, and for predicting the enzymatic cost of producing highly-
valued chemical compounds.
References:
1 D. McCloskey, B. Ø. Palsson and A. M. Feist, Mol. Syst. Biol., 2013, 9, 661.
2 R. Adadi, B. Volkmer, R. Milo, M. Heinemann and T. Shlomi, PLoS Comput. Biol., 2012, 8.
Biotechnology
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3 N. E. Lewis, H. Nagarajan and B. Ø. Palsson, Nat. Rev. Microbiol., 2012, 10, 291–305.
Glycerol as a substrate for mixotrophic microalgal production – identifying high performing strains and the effect on growth and
biochemical composition
Oscar Svensson, Joshua Mayers, Eva Albers
Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Goteborg
The production of various high-value compounds from photosynthetic microalgae
is receiving increasing attention as a sustainable alternative to many industrial and
agricultural processes1. However, achieving high and consistent growth rates is a
potential barrier to the realisation of these technologies. Growth of microalgae on
organic carbon sources in the presence of light (mixotrophy/photoheterotrophy)
is a possible strategy believed to improve biomass yields and increase
productivities. However, the use of conventional carbon sources such as glucose
is associated with several issues regarding competition with food production and
its affordability on a large scale. Due to expansions in biodiesel production, the
residual glycerol is now increasingly abundant and affordable2, and may represent
a suitable carbon source for microalgal growth. Cultivation on glycerol and its
effect on biochemical and metabolic pathways are little understood in microalgae
and warrants further investigation both from fundamental and applied standpoints.
This work seeks to identify mixotrophic green microalgal strains from the
Scenedesmus related-genera capable of high growth rates and biomass
productivities, and subsequently investigate the effect on biochemical and
metabolite composition. The production of high-value compounds, specifically
antioxidant carotenoids is of particular interest. The effects of possible
contaminants (methanol, salts, etc.) in crude glycerol on growth and biochemical
composition of productive strains are also assessed. Insights into the metabolism
of glycerol of microalgae will also be presented. These results will be considered
with the perspective of developing a feasible, future biorefinery scenario
successfully integrated with several industrial sectors.
References.
1) Wijffels, et al., 2010. Microalgae for the production of bulk chemicals and biofuels.
Biofuel. Bioprod. Bioref. 4: 287-295.
2) Li, et al., 2013. Microbial conversion of waste glycerol from biodiesel production into
value-added products. Energies 6
Biotechnology
105
3D bioprinting of chondrocytes for cartilage regeneration with the use of CELLINKTM
Ivan Tournierb, Kajsa Markstedtb, Athanasios Mantasb, Daniel Häggb and Paul Gatenholma
a Wallenberg Wood Science Center b Biopolymer Technology, Department of Chemistry and Chemical Engineering
3D bioprinting is an early emerging technology expected to revolutionize the field
of tissue engineering and regenerative medicine, which enables the reconstruction
of living tissue and organs preferably using the patient’s own cells. The 3D
bioprinter is a robotic arm able to move in the X,Y,Z directions with a resolution
of 10µm while dispensing fluids. The 3D bioprinter can position several cell types
and thus reconstruct the architecture of complex organs. This project aims at
developing a new supporting material, CELLINKTM, for printing living tissue
with cells and more precisely cartilage. CELLINKTM is composed of a
nanofibrillated cellulose dispersion and alginate. The structure can be crosslinked
after printing offering the printed tissue good stiffness and robustness. We have
compared different compositions of CELLINKTM and nanocellulose fibrils from
different sources, such as wood and bacteria. The rheology of the inks shows shear
thinning behavior and depending on their viscosity different parameters are
required for optimizing the printing resolution: pressure, needle diameter, printing
speed, temperature, etc. Cytotoxicity and cell viability have been tested in order
to print CELLINKTM with living chondrocytes.
Printing of CELLINK scaffold with 3D bioprinted meniscus
Regenhu Bioprinter 3D Discovery® of sheep with chondrocytes
References:
Markstedt, K.; Mantas, A.; Tournier, I.; Martínez Ávila, H.; Hägg, D.; Gatenholm, P., 3D Bioprinting Human
Chondrocytes with Nanocellulose–Alginate Bioink for Cartilage Tissue Engineering Applications.
Biomacromolecules 2015
Biotechnology
106
Screening of marine microalgae suitable as a potential feedstock for green chemical production
Sigita Vaiciulytea, Joshua Mayersa, Anna Godheb, Susanne Ekendahlc
Eva Albersa
aDept. of Biology and Biological engineering, Division of industrial Biotechnology, Chalmers University of Technology, Sweden,
bDept. of Biological and Environmental Sciences, Gothenburg University, Sweden, cDept. of Chemistry, Materials and Surfaces, SP Technical Research Institute of
Sweden
E-mail: [email protected]
Microalgae are considered as a potential viable feedstock for high-value industrial
chemical production due to the advantages of high growth rate in comparison with
terrestrial plants, efficient carbon dioxide fixation, and not competing for arable
lands and potable water. Microalgal polysaccharides can potentially be converted
into fermentable sugars for subsequent ethanol, butanol and acetone production
via microbial fermentation by e.g. yeasts or Clostridium sp.
Microalgal carbohydrates are the major products derived from carbon fixation
metabolism (i.e. the Calvin cycle) via photosynthesis. These carbohydrates are
accumulated either in the plastid thylakoids as reserve materials such as starch, or
become components of cell wall as cellulose, pectin, and sulfated polysaccharides.
However, the metabolism and composition of microalgal carbohydrates may
differ significantly from species to species and under different growth conditions.
Thus in this study, the main focus is the selection of fast-growing marine
microalgal strains with high carbohydrate content. Development of strategies to
improve biomass and carbohydrate productivity, as well as the sustainability of
cultivation processes, such as supplementation with carbon dioxide rich flue-gas,
will also be considered.
References:
1. Chen C.Y., Zhao X.Q.,Yen H.W., Hod S.H., Cheng C.L., Lee D. J., Bai F.W., Chang J.S. (2013).
Microalgae-based carbohydrates for biofuel production. Biochemical Engineering Journal, 78: 1-10
2. Ellis J. T., Hengge N.N., Sims R.C., Miller C.D. (2012). Acetone, butanol, and ethanol production
from wastewater algae. Bioresource Technology Journal. 111: 491-495
3. Ho S.H., Huang S.W., Chen C.Y., Hasunuma T., Kondo A., Chang J.C. (2013). Characterization and
optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E.
Bioresource Technology Journal. 135: 157-165
Biotechnology
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Process optimization and scale-up of fed-batch simultaneous saccharification and co-fermentation for efficient production of
lignocellulosic ethanol
Ruifei Wang, Johan Westman, Pornkamol Unrean, Lisbeth Olsson and Carl Johan Franzén
Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Göteborg, Sweden
Email: [email protected]
Fed-batch simultaneous saccharification and co-fermentation (SSCF) enables
production of lignocellulosic ethanol with high content of water insoluble solids
(WIS), and therefore high cellulose loadings (the major sugar source in
lignocellulose). The viscosity of the SSCF broth and the mass/heat transfer
efficiency, depend on the feeding frequency of solid substrates and the hydrolytic
activities of the added cellulases. An ideal feeding scheme should avoid over-feed
which leads to mixing problems, while feeding in as much substrates as possible
to shorten the process time and increase the final ethanol titer.
A previously developed kinetic model [1] was modified to predict the
performance of cellulases on steam pre-treated wheat straw, and to decide when
and how much WIS to feed in the next feeding event. With this approach, mixing
problems could be completely avoided up to 22.2% WIS, and ethanol
concentrations reached 56 g/L within 72 hours of SSCF. The process was tested
at demonstration scale in 10 m3 reactors, and a similar fermentation performance
as that in lab scale was observed.
Further feeding of solid substrate (>20% WIS) did not lead to increases in the
ethanol concentration, while a substantial loss of yeast viability (colony forming
unit) were observed in SSCF medium at high WIS contents. This was likely due
to toxic compounds retained in the pre-treated lignocellulose. We are currently
testing different xylose fermenting Saccharomyces cerevisiae strains in SSCF
process to investigate the possibilities to increase the ethanol titer further.
References.
1. Wang et al., 2014 Bioresour. Technol.
Biotechnology
108
Scale-up of multi feed fed-batch simultaneous saccharification and co-fermentation of pretreated wheat straw to ethanol
Johan O. Westman, Ruifei Wang, Pornkamol Unrean, Lisbeth Olsson and Carl Johan Franzén
Biology and Biological Engineering – Industrial biotechnology, Chalmers University of Technology, 412 96 Göteborg, Sweden.
Email: [email protected]
A major remaining issue with second-generation bioethanol production is the
difficulty of reaching high enough titers to facilitate an overall economical
process. Utilization of approximately 20% pretreated insoluble lignocellulosic
material in the process is necessary to reach an often mentioned ethanol
concentration of 4-5% (w/w). The viscosity at this solids concentration becomes
higher than what is easily attainable in most reactor set-ups. We have designed a
fed-batch simultaneous saccharification and co-fermentation (SSCF) process for
ethanol production from pretreated wheat straw up to 21% water insoluble solids
in a stirred tank reactor. In addition to feeding of solids at different time points,
feeding of fresh cells at different time points was found to be beneficial for the
process. The fed cells were adapted to the toxic environment by pre-cultivation in
the liquid fraction from the pretreatment. Enzyme addition at different time points
did however not improve the process, compared to addition of the same total
amount in the beginning of the fed-batch. The effectiveness of the optimized
process has been proven at demonstration scale in a 10 m3 SSCF reactor, reaching
ethanol concentrations of 5% (w/w). A further increase was hindered by the
toxicity of the medium, lowering the cells’ fermentation capacity. We have
previously shown that strong flocculation can increase the ability of yeast to
ferment toxic lignocellulose hydrolysates [1]. We therefore created strongly
flocculating xylose fermenting Saccharomyces cerevisiae strains and are
currently investigating these in the SSCF process.
References.
Westman et al. Appl Environ Microbiol, 2014.
Biotechnology
109
Electrofermentations for efficient conversion of waste glycerol into value-added 1,3-propanediol
Nikolaos Xafeniasa and Valeria Mapellia
aDivision of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, SE-41296, Sweden
Email: [email protected]
The biodiesel industry has to face a great challenge regarding the treatment of
increasing volumes of glycerol produced annually as a by-product. An
economically viable solution is to convert glycerol into 1,3-propanediol (1,3-
PDO), a more valuable chemical used for the production of for example polymers,
lubricants, and cosmetics. Electrofermentations taking place in
bioelectrochemical systems could shift the pathways of conventional
fermentations, enhancing 1,3-PDO production. When a polarized cathode was
embedded in the fermentation, 1,3-PDO production from glycerol was greatly
enhanced (Figure 1a). Biofilms formed on the cathode electrodes improved the
1,3-PDO production rates more than five times, and 1,3-PDO concentrations
reached values up to 42 g 1,3-PDO/L. Sequencing of the 16S rDNA revealed how
the applied electric potential shifted the dominant bacterial families (Figure 1b),
in relation to the higher (electro)fermentation rates observed. Clostridiaceae,
together with Veillonellaceae, dominated the bioelectrochemical reactors, where
butyrate was also produced at high concentrations. In the absence of electrodes
lactate was the second main metabolite and Lactobacillaceae were predominant,
while 1,3-PDO was inefficiently produced. Our results demonstrate that the
biofilm has a very important role, and that applying the right electrochemical
conditions would help to enrich the right microbial consortia in favor of higher
1,3-PDO concentrations.
Figure 1. The effect of applied potential (-1.1 V vs. SHE) on 1,3-PDO production (a) and on bacterial
enrichment (b).
Biotechnology
110
Metabolic Engineering for Production of Oleo-chemicals and Advanced Biofuels in Yeast
Yongjin Zhoua, Nicolaas A. Buijsa, Zhiwei Zhua, Verena Siewersa, Jens Nielsena
aDepartment of Biology and Biological Engineering, Chalmers University of Technology, Sweden
Email: [email protected]
Volatile energy costs and pressure to conserve fossil fuel resources have ignited
efforts to produce biofuels and renewable commodity chemicals via microbial
fermentation of biomass. The fatty acid-based biofuels (fatty acids, alkanes, fatty
alcohol, etc.) are considered as ideal alternative to fossil based chemicals and
advanced biofuels. Though most of these molecules have been microbial
synthesized，the titers remains to be largely improved for industrial process. As
Saccharomyces cerevisiae is a well-studied industrial model microorganism and
the overall metabolism including the lipid metabolism is well studied, it is feasible
to engineer lipid metabolism for overproduction of oleo-molecules in S.
cerevisiae. Here, we multivariate engineered the lipid metabolism by increasing
the supply of precursor, inhibition of by-product formation, and disrupting the
reversal futile cycling, which enabled production of 2.4 g/L free fatty acid, the
highest titer in yeast. Then this fatty acid overproducing strain was tailored for
fatty alcohol biosynthesis by using a carboxylic acid reductase. After enhancing
the fatty aldehyde reduction by engineering alcohol dehydrogenation and
reduction, fatty alcohol production reached 0.52 g/L. This results provided many
interesting targets for further enhancing the production of oleo-chemicals and
advanced biofuels.
Biotechnology
111
Peroximes as a novel tool for pathway evaluation in S. cerevisiae
Paulo Gonçalves Teixeira, Yongjin Zhou, Verena Siewers and Jens Nielsen
Division of Systems and Synthetic Biology - Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
Email: [email protected]
Our studies in metabolic engineering have shown that production of fatty-acyl-
CoA and fatty acid derived products in S. cerevisiae at a lab scale can be increased
by targeting the enzymes responsible for this conversion to the peroxisomes
present in the cell. This use of an alternative organelle makes it a valuable tool in
the development phase of synthetic metabolic pathways since it provides
compartimentalized and efficient production of target molecules combined with a
specialized import of fatty acyl-CoAs and fatty acids which is relevant for
pathways that use these metabolites as percursors. However, the tight regulation
and physiological constrains of peroxisome biogenesis in Saccharomyces can
bring problems to the implementation of this tool when we want higher titers of
the product of interest.
In this study we screen a library of mutants that have single-deletions in genes
involved in peroxisome biogenesis and degradation. Each strain is transformed
with a peroxisome-targeted fatty acyl-CoA reductase and the fatty alcohol
productivity of each mutant is analyzed and compared to the wild-type. This way
we hope to understand the limiting factors and recognize targets that could allow
us to improve the productivity of this peroxisome-targeted pathway and as such
plan the development of efficient strains for evaluating peroxisome-targeted
pathways.
Biotechnology
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The impact of respiration and oxidative stress on recombinant α-amylase production by the model yeast Saccharomyces cerevisiae.
José L. Martínez*, Eugenio Meza, Dina Petranovic, Jens Nielsen.
Systems and Synthetic Biology, Dept. Chemical and Biological Engineering. Chalmers University of Technology. Kemivägen 10, SE-41296 Göteborg (Sweden).
*[email protected] Abstract The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of either therapeutic or industrial interest. However, heterologous protein productivity is often low due to limitations of the selected host strain (1). In the case of the α-amylase, overproduction by S. cerevisiae may lead to
an intracellular accumulation of reactive oxygen species (ROS) as a consequence of the formation of disulfide bonds during protein folding in the endoplasmic reticulum, and therefore causing a significant decrease in the cell performance which ultimately affects the protein titer and productivity (2). In order to reduce the internal ROS levels, we describe a strategy based on the overexpression of the Hap1 transcription factor, known to be responsible for both the activation of a set of genes involved in ROS detoxification and the oxidative stress response, as well as genes related to the respiratory metabolism (3). This strategy resulted to have a positive effect in the protein production by increasing the final titer compared to the control strains, while keeping the ROS levels constant during batch cultivations.
1. Hou J, Osterlund T, Liu Z, Petranovic D, Nielsen J. 2013. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 97:3559-3568.
2. Liu Z, Osterlund T, Hou J, Petranovic D, Nielsen J. 2013. Anaerobic alpha-amylase production and secretion with fumarate as the final electron acceptor in Saccharomyces cerevisiae. Appl Environ Microbiol 79:2962-2967.
3. Zhang L, Hach A. 1999. Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator. Cell Mol Life Sci 56:415-426.
