Kaisa Karhumaa

Bio: Kaisa Karhumaa is an academic researcher from Lund University. The author has contributed to research in topics: Xylose & Fermentation. The author has an hindex of 17, co-authored 22 publications receiving 2941 citations. Previous affiliations of Kaisa Karhumaa include Technical University of Denmark & Carlsberg Laboratory.

Towards industrial pentose-fermenting yeast strains

Abstract: Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

Metabolic Engineering for Pentose Utilization in Saccharomyces cerevisiae

Abstract: The introduction of pentose utilization pathways in baker's yeast Saccharomyces cerevisiae is summarized together with metabolic engineering strategies to improve ethanolic pentose fermentation. Bacterial and fungal xylose and arabinose pathways have been expressed in S. cerevisiae but do not generally convey significant ethanolic fermentation traits to this yeast. A large number of rational metabolic engineering strategies directed among others toward sugar transport, initial pentose conversion, the pentose phosphate pathway, and the cellular redox metabolism have been exploited. The directed metabolic engineering approach has often been combined with random approaches including adaptation, mutagenesis, and hybridization. The knowledge gained about pentose fermentation in S. cerevisiae is primarily limited to genetically and physiologically well-characterized laboratory strains. The translation of this knowledge to strains performing in an industrial context is discussed.

A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance.

Abstract: The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity.

Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

Abstract: Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering

Abstract: A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae. Copyright (c) 2005 John Wiley & Sons, Ltd. (Less)

Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production

Abstract: Biofuels produced from various lignocellulosic materials, such as wood, agricultural, or forest residues, have the potential to be a valuable substitute for, or complement to, gasoline. Many physicochemical structural and compositional factors hinder the hydrolysis of cellulose present in biomass to sugars and other organic compounds that can later be converted to fuels. The goal of pretreatment is to make the cellulose accessible to hydrolysis for conversion to fuels. Various pretreatment techniques change the physical and chemical structure of the lignocellulosic biomass and improve hydrolysis rates. During the past few years a large number of pretreatment methods have been developed, including alkali treatment, ammonia explosion, and others. Many methods have been shown to result in high sugar yields, above 90% of the theoretical yield for lignocellulosic biomasses such as woods, grasses, corn, and so on. In this review, we discuss the various pretreatment process methods and the recent literature that...

Bioconversion of lignocellulose: inhibitors and detoxification

Abstract: Bioconversion of lignocellulose by microbial fermentation is typically preceded by an acidic thermochemical pretreatment step designed to facilitate enzymatic hydrolysis of cellulose. Substances formed during the pretreatment of the lignocellulosic feedstock inhibit enzymatic hydrolysis as well as microbial fermentation steps. This review focuses on inhibitors from lignocellulosic feedstocks and how conditioning of slurries and hydrolysates can be used to alleviate inhibition problems. Novel developments in the area include chemical in-situ detoxification by using reducing agents, and methods that improve the performance of both enzymatic and microbial biocatalysts.