Robert Mans

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.

A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.

Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

Glycerol production by Saccharomyces cerevisiae, which is required for redox‐cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol‐3‐phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF‐expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid‐borne mhpF gene for anaerobic growth.

Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production

Evolutionary engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, evolutionary engineering has also become a powerful approach for identification and reverse engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of evolutionary engineering to products of yeasts whose synthesis requires a net input of cellular energy.

An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake‐flask cultures. A combination of RNAseq‐based transcriptome analysis and 13C‐15N‐based flux analysis was used to elucidate the arginase‐independent pathway. Isotopic 13C15N‐enrichment in γ‐aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid

Abstract While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade− mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

Abstract Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose‐based processes remain to be investigated. An essential first step is to identify all native sucrose‐hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose‐negative strains. A strain in which all known sucrose‐transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose‐hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h−1 after sequential batch cultivation. This increase was accompanied by a 3‐fold increase of in vitro sucrose‐hydrolysis and isomaltase activities, as well as by a 3‐ to 5‐fold upregulation of the isomaltase‐encoding genes IMA1 and IMA5. One‐step Cas9‐mediated deletion of all isomaltase‐encoding genes (IMA1‐5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose‐negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport.

Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae

Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02h-1 (LmSPase) and 0.06 ± 0.01h-1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01h-1 and 0.08 ± 0.00h-1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.

Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae

NAD(P)‐malic enzyme (NAD(P)‐ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2, and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP‐ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD‐dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)‐ME isoforms, as well as the NADP‐ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP‐ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase‐negative (Pyc−) Saccharomyces cerevisiae strains. The heterologous expression of NADP‐ME1, NADP‐ME2 (and its mutant proteins), and NADP‐ME3 restored the growth of Pyc− S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2. On the other hand, NADP‐ME4, NAD‐ME1, and NAD‐ME2 could not rescue the Pyc− strains from C4 auxotrophy. NADP‐ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP‐ME, where this activity was correlated with the amount of NADP‐ME2 transcript. These results indicate that specific A. thaliana NADP‐ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP‐ME, which may contribute to the synthesis of C4 compounds and redox shuttling in plant cells.

Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgarisSuf1 transporter

Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1‐dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (μ = 0.19 h−1 and μ = 0.08 h−1, respectively). All five mutants displayed sucrose‐induced proton uptake (13–50 μmol H+ (g biomass)−1 min−1). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1I209F C265F G326C) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h−1) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1s transport mechanism.

Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics

Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous ‘Materials and Methods’ sections in scientific journals, thus promoting reproducibility and reusability of published studies.