Abstract: Antimetabolites, which are structural analogues of metabolites or carbon sources that exhibit toxicity by disrupting metabolism, can couple phenotypes of interest to growth and thereby facilitate the selection of relevant strains from large mutant pools generated by random mutagenesis. Toxicity and high costs of many antimetabolites provide incentives to minimise liquid volume during cultivation, while still capturing the genetic diversity in large mutant pools. This study aims to explore the use of high-throughput microbead-based cultivation and label-free enrichment of antimetabolite-resistant mutants. The generation of a large number (~ 10⁷ microbeads) of picolitre-sized cultivation compartments using water-in-oil emulsions enables high-throughput, parallel growth of millions of individual mutants in a total medium volume of just 0.3 mL. A reduction in screening medium volume reduces the quantities of expensive antimetabolites required. Employing a fluorescence-activated microbead-sorting-based approach enables label-free screening and selection of mutants based on autofluorescence signals as indicators for growth. The potential of this microbead-based, label-free selection method was demonstrated by enriching a G418 (geneticin) antibiotic-resistant and a glucose analogue (2-deoxyglucose) resistant Saccharomyces cerevisiae strains by 16,000-fold and 600-fold, respectively, in single-step enrichment experiments. Key points: • Small-volume cultivation cuts screening costs for costly antimetabolite resistance. • Autofluorescence-based screening enables label-free detection of resistant mutants. • 10⁴- and 10³-fold enrichment of antibiotic- and glucose analogue-resistant strains.

Current industrial processes for yeast-based conversion of glucose, xylose, and arabinose mixtures in lignocellulosic hydrolysates mostly rely on monocultures of generalist strains that consume the sugars sequentially. Co-cultivation of "sugar-specialist" strains offers potential advantages regarding volumetric productivity and genetic stability of pentose-fermenting strains during prolonged operation. However, in these cultures, the slow fermentation of pentoses strongly affects the time required for complete sugar conversion. This study explores whether the kinetics of such co-cultures can be improved by increasing the biomass concentration through selective sedimentation of a slow-fermenting strain in sequential batch fermentations. Deletion of ACE2 in a diploid, arabinose-specialist Saccharomyces cerevisiae strain yielded a multicellular, fast-sedimenting phenotype. In monocultures on 20 g∙L-1 arabinose in anaerobic sequential batch bioreactors with a 30-min biomass settling period between cultivation cycles, the sedimenting strain showed a shorter time for full conversion than its non-sedimenting parental strain (11.5 ± 0.4 h and 16.6 ± 0.4 h, respectively). Co-cultures of a glucose-specialist S. cerevisiae strain with the sedimenting arabinose specialist on a glucose-arabinose mixture (10 g·L-1 of each sugar) showed a 29% shorter batch time than corresponding co-cultures with the non-sedimenting arabinose specialist (12.6 ± 0.1 h vs. 17.7 ± 0.1 h). Increasing the biomass settling period to 1 h further reduced the batch time to 8.1 ± 0.1 h and resulted in near-simultaneous depletion of both sugars. These results show that if selective sedimentation of specific slow-fermenting strains can be implemented under relevant process conditions, it has the potential to improve microbial conversion of mixed substrates.IMPORTANCEFermentation of substrate mixtures from lignocellulosic biomass can reduce substrate costs but is often hindered by inefficient, sequential consumption of sugars by "sugar-generalist" Saccharomyces cerevisiae strains. While co-cultures of "sugar-specialist" S. cerevisiae strains can enable parallel consumption in sequential-batch fermentations, the overall volumetric productivity is limited by the slowest fermenter. This study presents a strategy to overcome this limitation by engineering a slow-fermenting arabinose-specialist S. cerevisiae strain for fast sedimentation and implementing a short settling phase between batch cycles in sequential-batch bioreactors, selectively concentrating the specialist's biomass and improving consumption kinetics in subsequent cycles. This approach reduced overall fermentation time by 30% in monoculture and enabled near-simultaneous depletion of arabinose and glucose in co-culture with a glucose-specialist strain. These results show that selective sedimentation of co-culture partners can improve fermentation kinetics in sequential-batch cultures. If compatible with industrial process conditions, this strategy has the potential to improve the economics of co-culture-based fermentations.

Large-scale microbial-biotechnology processes for production of chemicals almost exclusively rely on pure cultures of microbial strains. Especially for extensively engineered pure cultures, process performance can be negatively affected, which can be caused by issues such as pathway imbalance, deterioration of productivity caused by genetic instability and enzyme promiscuity. An increasing number of studies demonstrate that, under ‘academic’ laboratory conditions, the use of defined co-cultures (i.e. deliberate mixtures of known microbial strains) offers unique possibilities for mitigating such drawbacks. These advantages differ for dissimilatory products, whose synthesis from one or more carbon substrates provides cells with free energy, and assimilatory products, whose synthesis requires a net input of free energy. Based on advances in experimental and theoretical research, this paper highlights how defined co-cultures can address several limitations of mono-cultures for production of low-molecular-weight compounds. From this largely academic perspective, we outline the key challenges for scaling these systems to industry, which underscore the need for innovative solutions and continued research in this area.

Ethanol is an interesting renewable feedstock for microbial biotechnology. Compared to other yeasts, Cyberlindnera jadinii has an exceptionally high biomass yield on ethanol. Simulations with a stoichiometric metabolic model identified an intramitochondrial rather than cytosolic localization of NAD+-dependent alcohol and acetaldehyde dehydrogenases as a possible explanation for this high yield. This hypothesis was tested in a comparative study with Ogataea parapolymorpha, which, like C. jadinii, contains a proton-pumping Complex I NADH dehydrogenase, but shows a lower biomass yield on ethanol. Experiments with an aox null mutant showed that the lower biomass yield of O. parapolymorpha was not caused by in vivo alcohol oxidase activity. Proteome analyses showed that the most abundant ADH isozyme in C. jadinii and O. parapolymorpha contained and lacked a predicted mitochondrial targeting sequence, respectively. The ADH protein with the highest prevalence in ethanol-limited cultures of C. jadinii was predicted to be mitochondrial and accounted for almost 4% of the detected proteome. In subcellular fractionation studies with cells grown under ethanol limitation, ADH showed 10-fold lower specific activity in the mitochondria-enriched fraction of cell homogenates of O. parapolymorpha than in the cytosolic fraction. Conversely, C. jadinii showed a 50% higher ADH activity in the mitochondria-enriched fraction. A low overall recovery of ADH in mitochondria-enriched fractions of C. jadinii was attributed to disruption of the reticulate mitochondrial structures observed in ethanol-grown cells during preparation of cell homogenates. This study demonstrates how subcellular localization of an NAD+-dependent dehydrogenase can influence respiratory energy coupling in yeasts that harbor a Complex I NADH dehydrogenase. IMPORTANCE: This study provides theoretical and experimental evidence that intracellular localization of NAD+-dependent dehydrogenases can be a key factor for respiratory energy coupling in yeasts. Based on this, alteration of the cellular localization of NAD+-dehydrogenases is identified as an interesting metabolic engineering strategy for improving respiratory energy coupling in eukaryotic cell factories.

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h⁻¹ in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h⁻¹) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.

Emerging low-emission production technologies make ethanol an interesting substrate for yeast biotechnology, but information on growth rates and biomass yields of yeasts on ethanol is scarce. Strains of 52 Saccharomycotina yeasts were screened for growth on ethanol. The 21 fastest strains, among which representatives of the Phaffomycetales order were overrepresented, showed specific growth rates in ethanol-grown shake-flask cultures between 0.12 and 0.46 h⁻¹. Seven strains were studied in aerobic, ethanol-limited chemostats (dilution rate 0.10 h⁻¹). Saccharomyces cerevisiae and Kluyveromyces lactis, whose genomes do not encode Complex-I-type NADH dehydrogenases, showed biomass yields of 0.59 and 0.56 gbiomass g_ethanol⁻¹, respectively. Different biomass yields were observed among species whose genomes do harbour Complex-I-encoding genes: Phaffomyces thermotolerans (0.58 g g⁻¹), Pichia ethanolica (0.59 g g⁻¹), Saturnispora dispora (0.66 g g⁻¹), Ogataea parapolymorpha (0.67 g g⁻¹), and Cyberlindnera jadinii (0.73 g g⁻¹). Cyberlindnera jadinii biomass showed the highest protein content (59 ± 2%) of these yeasts. Its biomass yield corresponded to 88% of the theoretical maximum that is reached when growth is limited by assimilation rather than by energy availability. This study suggests that energy coupling of mitochondrial respiration and its regulation will become key factors for selecting and improving yeast strains for ethanol-based processes.

Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results: In slow-growing anaerobic chemostat cultures (D = 0.05 h ⁻¹), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h ⁻¹ but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h ⁻¹) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h ⁻¹, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower. Conclusions: Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

In anaerobic Saccharomyces cerevisiae cultures, NADH (reduced form of nicotinamide adenine dinucleotide)-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as 'redox-sink' and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation. An alternative glycerol-reduction strategy bypasses the oxidative reaction in glycolysis by introducing phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). For optimal performance in industrial settings, yeast strains should ideally first fully convert acetate and, subsequently, continue low-glycerol fermentation via the PRK-RuBisCO pathway. However, anaerobic batch cultures of a strain carrying both pathways showed inferior acetate reduction relative to a strain expressing only the A-ALD pathway. Complete A-ALD-mediated acetate reduction by a dual-pathway strain, grown anaerobically on 50 g L-1 glucose and 5 mmol L-1 acetate, was achieved upon reducing PRK abundance by a C-terminal extension of its amino acid sequence. Yields of glycerol and ethanol on glucose were 55% lower and 6% higher, respectively, than those of a nonengineered reference strain. The negative impact of the PRK-RuBisCO pathway on acetate reduction was attributed to sensitivity of the reversible A-ALD reaction to intracellular acetaldehyde concentrations.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the Saccharomyces cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.

Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast Saccharomyces cerevisiae. This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth and glycerol formation. Particular attention is paid to strategies aimed at altering energy coupling of alcoholic fermentation and to strategies for altering redox-cofactor coupling in carbon and nitrogen metabolism that aim to reduce or eliminate the role of glycerol formation in anaerobic redox metabolism. In addition to providing an overview of scientific advances we discuss context dependency, theoretical impact and potential for industrial application of different proposed and developed strategies.

Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the Saccharomyces cerevisiae Class-I dihydroorotate dehydrogenase Ura1, which supports synthesis of pyrimidines under aerobic and anaerobic conditions. However, expression of codon-optimised representatives of this gene family, from the ascomycete Alternaria alternata and the basidiomycete Schizophyllum commune, only supported growth of an S. cerevisiae ura1Δ mutant when synthetic media were supplemented with dihydrouracil. A hypothesis that these genes encode NAD(P)⁺-dependent dihydrouracil dehydrogenases (EC 1.3.1.1 or 1.3.1.2) was rejected based on absence of complementation in anaerobic cultures. Uracil- and thymine-dependent oxygen consumption and hydrogen-peroxide production by cell extracts of S. cerevisiae strains expressing the A. alternata and S. commune genes showed that, instead, they encode active dihydrouracil oxidases (DHO, EC1.3.3.7). DHO catalyses the reaction dihydrouracil + O₂ → uracil + H₂O₂ and was only reported in the yeast Rhodotorula glutinis (Owaki in J Ferment Technol 64:205–210, 1986). No structural gene for DHO was previously identified. DHO-expressing strains were highly sensitive to 5-fluorodihydrouracil (5F-dhu) and plasmids bearing expression cassettes for DHO were readily lost during growth on 5F-dhu-containing media. These results show the potential applicability of fungal DHO genes as counter-selectable marker genes for genetic modification of S. cerevisiae and other organisms that lack a native DHO. Further research should explore the physiological significance of this enigmatic and apparently widespread fungal enzyme.

Background: Saccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis. Results: Consistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD⁺-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high K_m for CO₂ of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO₂. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway. Conclusions: Combination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

Following publication of the original article [1], the authors reported errors in the text of the Results section and in Table 2. It refers to a mutation in a yeast gene as VPS1^I410L and to the corresponding change in the Vps1 amino-acid sequence as I410L. The correct descriptions should read VPS1^I401L and I401L, respectively. This has been corrected with this erratum.