Reductive amination is one of the most synthetically direct routes to access chiral amines. Several Imine Reductases (IREDs) have been discovered to catalyze reductive amination (Reductive Aminases or RedAms), yet they are dependent on the expensive phosphorylated nicotinamide adenine dinucleotide cofactor NADPH and usually more active at basic pH. Here, we describe the discovery and synthetic potential of an IRED from Rhodococcus erythropolis (RytRedAm) that catalyzes reductive amination between a series of medium to large carbonyl and amine compounds with conversions of up to >99% and 99% enantiomeric excess at neutral pH. RytRedAm catalyzes the formation of a substituted γ-lactam and N-methyl-1-phenylethanamine with stereochemistry opposite to that of fungal RedAms, giving the (S)-enantiomer. This enzyme remarkably uses both NADPH and NADH cofactors with K_M values of 15 and 247 μM and turnover numbers k_cat of 3.6 and 9.0 s^-1, respectively, for the reductive amination of hexanal with allylamine. The crystal structure obtained provides insights into the flexibility to also accept NADH, with residues R35 and I69 diverging from that of other IREDs/RedAms in the otherwise conserved Rossmann fold. RytRedAm thus represents a subfamily of enzymes that enable synthetic applications using NADH-dependent reductive amination to access complementary chiral amine products.

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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.

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Background
Elimination of greenhouse gas emissions in industrial biotechnology requires replacement of carbohydrates by alternative carbon substrates, produced from CO2 and waste streams. Ethanol is already industrially produced from agricultural residues and waste gas and is miscible with water, self-sterilizing and energy-dense. The yeast C. jadinii can grow on ethanol and has a history in the production of single-cell protein (SCP) for feed and food applications. To address a knowledge gap in quantitative physiology of C. jadinii during growth on ethanol, this study investigates growth kinetics, growth energetics, nutritional requirements, and biomass composition of C. jadinii strains in batch, chemostat and fed-batch cultures.

Results
In aerobic, ethanol-limited chemostat cultures, C. jadinii CBS 621 exhibited a maximum biomass yield on ethanol (Y max X/S) of 0.83 gbiomass (gethanol)−1 and an estimated maintenance requirement for ATP (mATP) of 2.7 mmolATP (gbiomass)−1 h−1. Even at specific growth rates below 0.05 h−1, a stable protein content of approximately 0.54 gprotein (gbiomass)−1 was observed. At low specific growth rates, up to 17% of the proteome consisted of alcohol dehydrogenase proteins, followed by aldehyde dehydrogenases and acetyl-CoA synthetase. Of 13 C. jadinii strains evaluated, 11 displayed fast growth on ethanol (μmax > 0.4 h−1) in mineral medium without vitamins, and CBS 621 was found to be a thiamine auxotroph. The prototrophic strain C. jadinii CBS 5947 was grown on an inorganic salts medium in fed-batch cultures (10-L scale) fed with pure ethanol. Biomass concentrations in these cultures increased up to 100 gbiomass (kgbroth)−1, with a biomass yield of 0.65 gbiomass (gethanol)−1. Model-based simulation, based on quantitative parameters determined in chemostat cultures, adequately predicted biomass production. A different protein content of chemostat- and fed-batch-grown biomass (54 and 42%, respectively) may reflect the more dynamic conditions in fed-batch cultures.

Conclusions
Analysis of ethanol-grown batch, chemostat and fed-batch cultures provided a quantitative physiology baseline for fundamental and applied research on C. jadinii. Its high maximum growth rate, high energetic efficiency of ethanol dissimilation, simple nutritional requirements and high protein content, make C. jadinii a highly interesting platform for production of SCP and other products from ethanol.@en

The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.PK-derived Saccharomyces cerevisiae SpyCas9 expressing strain (IMX2600), that serve as chassis of new cell-factories.

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Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.

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Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.

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Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for the enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification of spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as the sole carbon source, revealing the impact of growth conditions on mitochondrial RNA expression and splicing. This study uncovered a remarkable difference in the turnover of Group II introns between yeast grown in either mostly fermentative or fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to monitor mitochondrial transcriptome responses in a broad range of relevant contexts, including oxidative stress, apoptosis and mitochondrial diseases.

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The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

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Background: In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results: Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions: Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

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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.

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An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.

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Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.

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The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular neochromosomes of 50 and 100 kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. Neochromosomes made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding neochromosomes were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

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Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN. PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.

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Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD⁺-dependent oxidation of 10-hydroxystearic acid.

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Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

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The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

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BACKGROUND: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

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The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separate order of alkaliphilic bacteria closely related to the Bacillales order. Efforts to relate the genomic information of this evolutionary ancient organism to environmental adaptation have been thwarted by the inability to construct a complete genome. The existing draft genome is highly fragmented due to repetitive regions, and gaps between and over repetitive regions were unbridgeable. To address this, Oxford Nanopore Technology’s MinION allowed us to span these repeats through long reads, with over 6000-fold coverage. This resulted in a single 3.34 Mb circular chromosome. The profile of transporters and central metabolism gives insight into why the organism prefers glutamate over sucrose as carbon source. We propose that the deamination of glutamate allows alkalization of the immediate environment, an excellent example of how an extremophile modulates environmental conditions to suit its own requirements. Curiously, plant-like hallmark electron transfer enzymes and transporters are found throughout the genome, such as a cytochrome b₆c₁ complex and a CO₂-concentrating transporter. In addition, multiple self-splicing group II intron-encoded proteins closely aligning to those of a telomerase reverse transcriptase in Arabidopsis thaliana were revealed. Collectively, these features suggest an evolutionary relationship to plant life.

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Engineering living cells for production of chemicals, enzymes and therapeutics can burden cells due to use of limited native co-factor availability and/or expression burdens, totalling a fitness deficit compared to parental cells encoded through long evolutionary trajectories to maximise fitness. Ultimately, this discrepancy puts a selective pressure against fitness-burdened engineered cells under prolonged bioprocesses, and potentially leads to complete eradication of high-performing engineered cells at the population level. Here we present the mutation landscapes of fitness-burdened yeast cells engineered for vanillin-β-glucoside production. Next, we design synthetic control circuits based on transcriptome analysis and biosensors responsive to vanillin-β-glucoside pathway intermediates in order to stabilize vanillin-β-glucoside production over ~55 generations in sequential passage experiments. Furthermore, using biosensors with two different modes of action we identify control circuits linking vanillin-β-glucoside pathway flux to various essential cellular functions, and demonstrate control circuits robustness and almost 2-fold higher vanillin-β-glucoside production, including 5-fold increase in total vanillin-β-glucoside pathway metabolite accumulation, in a fed-batch fermentation compared to vanillin-β-glucoside producing cells without control circuits.

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