James C. du Preez

The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review

Alcohol dehydrogenases (ADHs) constitute a large family of enzymes responsible for the reversible oxidation of alcohols to aldehydes with the concomitant reduction of NAD(+) or NADP(+). These enzymes have been identified not only in yeasts, but also in several other eukaryotes and even prokaryotes. The ADHs of Saccharomyces cerevisiae have been studied intensively for over half a century. With the ever-evolving techniques available for scientific analysis and since the completion of the Yeast Genome Project, a vast amount of new information has been generated during the past 10 years. This review attempts to provide a brief summary of the wealth of knowledge gained from earlier studies as well as more recent work. Relevant aspects regarding the primary and secondary structure, kinetic characteristics, function and molecular regulation of the ADHs in S. cerevisiae are discussed in detail. A brief outlook also contemplates possible future research opportunities.

The effects of the novel bifidogenic trisaccharide, neokestose, on the human colonic microbiota

The potential prebiotic effect of the fructo-trisaccharide, neokestose, on intestinal bacteria was investigated. Bifidobacterium sp. utilized neokestose to a greater extend and produced more biomass from neokestose than facultative anaerobes under anaerobic conditions in batch culture. Lactobacillus salivarius utilized glucose but negligible amounts of neokestose. L. salivarius and the facultative anaerobes produced significantly more biomass from glucose than from neokestose, whereas the biomass yields obtained with bifidobacteria on neokestose and glucose, respectively, were not significantly different. Static batch cultures inoculated with faeces supported the prebiotic effect of neokestose, which had been observed in the pure culture investigations. Bifidobacteria and lactobacilli were increased while potentially detrimental coliforms, clostridia and bacteroides, decreased after 24 h fermentation with neokestose. In addition, this effect was more pronounced with neokestose than with a commercial prebiotic fructo-oligosaccharide. It was concluded that neokestose has potential as a novel bifidogenic substance and that it might have advantages over the commercially available sources currently used.

Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism of Saccharomyces cerevisiae

The physiological role and possible functional substitution of each of the five alcohol dehydrogenase (Adh) isozymes in Saccharomyces cerevisiae were investigated in five quadruple deletion mutants designated strains Q1-Q5, with the number indicating the sole intact ADH gene. Their growth in aerobic batch cultures was characterised in terms of kinetic and stoichiometric parameters. Cultivation with glucose or ethanol as carbon substrate revealed that Adh1 was the only alcohol dehydrogenase capable of efficiently catalysing the reduction of acetaldehyde to ethanol. The oxidation of produced or added ethanol could also be attributed to Adh1. Growth of strains lacking the ADH1 gene resulted in the production of glycerol as a major fermentation product, concomitant with the production of a significant amount of acetaldehyde. Strains Q2 and Q3, expressing only ADH2 or ADH3, respectively, produced ethanol from glucose, albeit less than strain Q1, and were also able to oxidise added ethanol. Strains Q4 and Q5 grew poorly on glucose and produced ethanol, but were neither able to utilise the produced ethanol nor grow on added ethanol. Transcription profiles of the ADH4 and ADH5 genes suggested that participation of these gene products in ethanol production from glucose was unlikely.

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146xa0U ml−1, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322xa0U ml−1 was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19xa0kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21xa0kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.

Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose

We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus.

Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae.

The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35xa0°C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6xa0gxa0l−1 were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70xa0% of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation.

Kinetics of growth and leukotoxin production by Mannheimia haemolytica in continuous culture.

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66xa0gxa0l−1 in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88xa0mmol glucose g−1xa0h−1, respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15xa0gxa0l−1 and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.

Protein enrichment of an Opuntia ficus-indica cladode hydrolysate by cultivation of Candida utilis and Kluyveromyces marxianus.

BACKGROUND The cladodes of Opuntia ficus-indica (prickly pear cactus) have a low protein content; for use as a balanced feed, supplementation with other protein sources is therefore desirable. We investigated protein enrichment by cultivation of the yeasts Candida utilis and Kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass. RESULTS Dilute acid pretreatment and enzymatic hydrolysis of sun-dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Even though K. marxianus had a much higher growth rate and utilized l-arabinose and d-galactose more completely than C. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. Yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains. CONCLUSIONS This K. marxianus strain was weakly Crabtree positive. Despite its low biomass yield, its performance compared well with C. utilis. This is the first report showing that the protein content and quality of O. ficus-indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of C. utilis and K. marxianus.

Editorial: chemicals and bioproducts from biomass.

© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. The current driving force for the development of “clean” technologies for the production of fuels and chemicals from renewable resources stems mainly from environmental concerns, which have overshadowed concerns regarding the depletion of fossil fuel reserves and the price instability of crude oil. To accommodate the increasing interest in biotechnology for the production of chemicals, Biotechnology for Biofuels (BfB) took the decision to widen its scope to also include articles on the biological production of chemicals and bioproducts derived from biomass and CO2. Whereas a large proportion of the articles in BfB still deal with the pretreatment, hydrolysis and bioconversion of lignocellulosic biomass to ethanol, the journal also has published a number of interesting articles related to bio-based chemicals and other bioproducts. This retrospective thematic issue assembles recent articles since 2015 to highlight these areas of research. An interesting observation when compiling this thematic issue was that of these 32 articles, 11 originated from China—by far the most from any one country. The topics covered by these selected articles are rather diverse, with feedstocks ranging from sugars to syngas (a mixture of CO, H2 and CO2), while the products include various chemicals and potential alternative biofuels such as acetate, n-butanol, 1-propanol, 1,2-propanediol, lipids, shortand medium-chain monocarboxylic acids and their corresponding alcohols, α-linolenic acid, succinic acid, glycolic acid, 3-hydroxypropionic acid, geraniol, chitin, isoprene, alkanes, 1-alkenes and biohythane (a mixture of biomethane and biohydrogen) produced from waste sludge using microbial electrolysis cells. The increasing number of articles dealing with the use of glycerol as feedstock for the production of bioproducts such as malic acid, 1,3-propanediol, 2,3-butanediol, triacylglycerides, erythritol and citric acid is encouraging, since valorisation of the huge amounts of crude glycerol generated as the main by-product of biodiesel production by transesterification of animal fats and plant oils has become imperative. Also included in this collection of articles is a review by Luo et al. [1] on the microbial synthesis of poly-γ-glutamic acid, a bio-based chemical that is already widely used in several industries and the production of which by microbial fermentation is deemed cost-effective. Apart from conventional bioprocesses such as anaerobic digestion and fermentation, a significant increase was noted in the number of articles involving metabolic engineering and also protein engineering aimed at the production of chemicals and alternative biofuels. The genetically tractable Escherichia coli still featured as the favoured host microorganism for metabolic engineering. Yao et al. [2] investigated the co-metabolism of glycerol and glucose to determine the metabolic potential of a genetically engineered E. coli strain as a platform for the production of biofuels and chemicals. For the production of geraniol, an acyclic monoterpene alcohol that finds application in the perfume and other industries, Liu et al. [3] overexpressed several key pathway genes in E. coli. The production of n-butanol, which has excellent qualities for an alternative fuel, was investigated by Saini et al. [4] through engineering of the central metabolism of E. coli. A novel approach to the biosynthesis of longchain 1-alkenes from low-cost triacylglycerols as a “dropin” biofuel is described in the article by Yan et al. [5] on the development of a tandem biotransformation process incorporating cell-free systems (purified enzymes or cellfree extracts) and engineered E. coli whole cells. Protein engineering of key enzymes offers the prospect of tailoring biofuel formulations to desired specifications [6]. This selection of articles includes reports on metabolic engineering also of other microorganisms aimed at a variety of bioproducts. Extensive engineering of the workhorse of industrial biotechnology, Saccharomyces Open Access Biotechnology for Biofuels

Reviewer acknowledgement 2015

© 2016 du Preez et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Biotechnology for Biofuels