LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

Metabolic flux enhancement from the translational fusion of terpene synthases is linked to terpene synthase accumulation.

The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), corresponding with increases of a similar magnitude in terpene synthase levels. Our data demonstrate that increased in vivo enzyme levels - resulting from improved expression and/or improved protein stability - is a major driver of catalytic enhancement from enzyme fusion.

Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol-type SLs.

Process Proteomics of Beer Reveals a Dynamic Proteome with Extensive Modifications.

Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nanoscale beer production. Samples included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production.

Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae.

Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L-1 in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L-1 in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L-1 in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth.

Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway.

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae.

Sesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1% biomass; moreover, the specific growth rate declined. In order to re-direct carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86% to ~100mgL-1. In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

Dynamic balancing of isoprene carbon sources reflects photosynthetic and photorespiratory responses to temperature stress.

The volatile gas isoprene is emitted in teragrams per annum quantities from the terrestrial biosphere and exerts a large effect on atmospheric chemistry. Isoprene is made primarily from recently fixed photosynthate; however, alternate carbon sources play an important role, particularly when photosynthate is limiting. We examined the relative contribution of these alternate carbon sources under changes in light and temperature, the two environmental conditions that have the strongest influence over isoprene emission. Using a novel real-time analytical approach that allowed us to examine dynamic changes in carbon sources, we observed that relative contributions do not change as a function of light intensity. We found that the classical uncoupling of isoprene emission from net photosynthesis at elevated leaf temperatures is associated with an increased contribution of alternate carbon. We also observed a rapid compensatory response where alternate carbon sources compensated for transient decreases in recently fixed carbon during thermal ramping, thereby maintaining overall increases in isoprene production rates at high temperatures. Photorespiration is known to contribute to the decline in net photosynthesis at high leaf temperatures. A reduction in the temperature at which the contribution of alternate carbon sources increased was observed under photorespiratory conditions, while photosynthetic conditions increased this temperature. Feeding [2-(13)C]glycine (a photorespiratory intermediate) stimulated emissions of [(13)C1-5]isoprene and (13)CO2, supporting the possibility that photorespiration can provide an alternate source of carbon for isoprene synthesis. Our observations have important implications for establishing improved mechanistic predictions of isoprene emissions and primary carbon metabolism, particularly under the predicted increases in future global temperatures.

Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae.

BACKGROUND: Engineering dynamic, environmentally- and temporally-responsive control of gene expression is one of the principle objectives in the field of synthetic biology. Dynamic regulation is desirable because many engineered functions conflict with endogenous processes which have evolved to facilitate growth and survival, and minimising conflict between growth and production phases can improve product titres in microbial cell factories. There are a limited number of mechanisms that enable dynamic regulation in yeast, and fewer still that are appropriate for application in an industrial setting. RESULTS: To address this problem we have identified promoters that are repressed during growth on glucose, and activated during growth on sucrose. Catabolite repression and preferential glucose utilisation allows active growth on glucose before switching to production on sucrose. Using sucrose as an activator of gene expression circumvents the need for expensive inducer compounds and enables gene expression to be triggered during growth on a fermentable, high energy-yield carbon source. The ability to fine-tune the timing and population density at which gene expression is activated from the SUC2 promoter was demonstrated by varying the ratio of glucose to sucrose in the growth medium. Finally, we demonstrated that the system could also be used to repress gene expression (a process also required for many engineering projects). We used the glucose/sucrose system to control a heterologous RNA interference module and dynamically repress the expression of a constitutively regulated GFP gene. CONCLUSIONS: The low noise levels and high dynamic range of the SUC2 promoter make it a promising option for implementing dynamic regulation in yeast. The capacity to repress gene expression using RNA interference makes the system highly versatile, with great potential for metabolic engineering applications.

Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities.

BACKGROUND: Predictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources (glucose, sucrose, galactose and ethanol) and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of "constitutive" and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter. RESULTS: The "constitutive" promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels (P(HXT7), P(SSA1) and P(ADH2)) varied in induction strength on non-glucose carbon sources (sucrose, galactose and ethanol); in contrast to the "constitutive" promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several "constitutive" promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift. CONCLUSIONS: The data provides a foundation for predictable and optimised control of gene expression levels on different carbon sources and throughout batch fermentation, including during and after the diauxic shift. This information can be applied for designing expression approaches to improve yields, rates and titres in yeast cell factories.

Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite.

BACKGROUND: High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade. Lycopene production is known to vary between wild-type Escherichia coli hosts, but the reasons behind this variation have never been fully elucidated. RESULTS: In an examination of six E. coli strains we observed that strains also differ in their capacity for increased lycopene production in response to metabolic engineering. A combination of genetic complementation, quantitative SWATH proteomics, and biochemical analysis in closely-related strains was used to examine the mechanistic reasons for variation in lycopene accumulation. This study revealed that rpoS, a gene previously identified in lycopene production association studies, exerts its effect on lycopene accumulation not through modulation of pathway flux, but through alteration of cellular oxidative status. Specifically, absence of rpoS results in increased accumulation of reactive oxygen species during late log and stationary phases. This change in cellular redox has no effect on isoprenoid pathway flux, despite the presence of oxygen-sensitive iron-sulphur cluster enzymes and the heavy redox requirements of the methylerythritol phosphate pathway. Instead, decreased cellular lycopene in the ΔrpoS strain is caused by degradation of lycopene in the presence of excess reactive oxygen species. CONCLUSIONS: Our results demonstrate that lycopene is not a reliable indicator of isoprenoid pathway flux in the presence of oxidative stress, and suggest that caution should be exercised when using lycopene as a screening tool in genome-wide metabolic engineering studies. More extensive use of systems biology for strain analysis will help elucidate such unpredictable side-effects in metabolic engineering projects.

Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants.

Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty.

Metabolic engineering of volatile isoprenoids in plants and microbes.

The chemical properties and diversity of volatile isoprenoids lends them to a broad variety of biological roles. It also lends them to a host of biotechnological applications, both by taking advantage of their natural functions and by using them as industrial chemicals/chemical feedstocks. Natural functions include roles as insect attractants and repellents, abiotic stress protectants in pathogen defense, etc. Industrial applications include use as pharmaceuticals, flavours, fragrances, fuels, fuel additives, etc. Here we will examine the ways in which researchers have so far found to exploit volatile isoprenoids using biotechnology. Production and/or modification of volatiles using metabolic engineering in both plants and microorganisms are reviewed, including engineering through both mevalonate and methylerythritol diphosphate pathways. Recent advances are illustrated using several case studies (herbivores and bodyguards, isoprene, and monoterpene production in microbes). Systems and synthetic biology tools with particular utility for metabolic engineering are also reviewed. Finally, we discuss the practical realities of various applications in modern biotechnology, explore possible future applications, and examine the challenges of moving these technologies forward so that they can deliver tangible benefits. While this review focuses on volatile isoprenoids, many of the engineering approaches described here are also applicable to non-isoprenoid volatiles and to non-volatile isoprenoids.

Escherichia coli W shows fast, highly oxidative sucrose metabolism and low acetate formation.

Sugarcane is the most efficient large-scale crop capable of supplying sufficient carbon substrate, in the form of sucrose, needed during fermentative feedstock production. However, sucrose metabolism in Escherichia coli is not well understood because the two most common strains, E. coli K-12 and B, do not grow on sucrose. Here, using a sucrose utilizing strain, E. coli W, we undertake an in-depth comparison of sucrose and glucose metabolism including growth kinetics, metabolite profiling, microarray-based transcriptome analysis, labelling-based proteomic analysis and (13)C-fluxomics. While E. coli W grew comparably well on sucrose and glucose integration of the omics, datasets showed that during growth on each carbon source, metabolism was distinct. The metabolism was generally derepressed on sucrose, and significant flux rearrangements were observed in central carbon metabolism. These included a reduction in the flux of the oxidative pentose phosphate pathway branch, an increase in the tricarboxylic acid cycle flux and a reduction in the glyoxylate shunt flux due to the dephosphorylation of isocitrate dehydrogenase. But unlike growth on other sugars that induce cAMP-dependent Crp regulation, the phosphoenol-pyruvate-glyoxylate cycle was not active on sucrose. Lower acetate accumulation was also observed in sucrose compared to glucose cultures. This was linked to induction of the acetate catabolic genes actP and acs and independent of the glyoxylic shunt. Overall, the cells stayed highly oxidative. In summary, sucrose metabolism was fast, efficient and led to low acetate accumulation making it an ideal carbon source for industrial fermentation with E. coli W.

Translational fusion of terpene synthases for metabolic engineering: Lessons learned and practical considerations.

The step catalyzed by terpene synthases is a well-recognized and significant bottleneck in engineered terpenoid bioproduction. Consequently, substantial efforts have been devoted towards increasing metabolic flux catalyzed by terpene synthases, employing strategies such as gene overexpression and protein engineering. Notably, numerous studies have demonstrated remarkable titer improvements by applying translational fusion, typically by fusing the terpene synthase with a prenyl diphosphate synthase that catalyzes the preceding step in the pathway. The main appeal of the translational fusion approach lies in its simplicity and orthogonality to other metabolic engineering tools. However, there is currently limited understanding of the underlying mechanism of flux enhancement, owing to the unpredictable and often protein-specific effects of translational fusion. In this chapter, we discuss practical considerations when engineering translationally fused terpene synthases, drawing insights from our experience and existing literature. We also provide detailed experimental workflows and protocols based on our previous work in budding yeast (Saccharomyces cerevisiae). Our intention is to encourage further research into the translational fusion of terpene synthases, anticipating that this will contribute mechanistic insights not only into the activity, behavior, and regulation of terpene synthases, but also of other enzymes.

ß-Dicarbonyls Facilitate Engineered Microbial Bromoform Biosynthesis.

Ruminant livestock produce around 24% of global anthropogenic methane emissions. Methanogenesis in the animal rumen is significantly inhibited by bromoform, which is abundant in seaweeds of the genus Asparagopsis. This has prompted the development of livestock feed additives based on Asparagopsis to mitigate methane emissions, although this approach alone is unlikely to satisfy global demand. Here we engineer a non-native biosynthesis pathway to produce bromoform in vivo with yeast as an alternative biological source that may enable sustainable, scalable production of bromoform by fermentation. ß-dicarbonyl compounds with low pKa values were identified as essential substrates for bromoform production and enabled bromoform synthesis in engineered Saccharomyces cerevisiae expressing a vanadate-dependent haloperoxidase gene. In addition to providing a potential route to the sustainable biological production of bromoform at scale, this work advances the development of novel microbial biosynthetic pathways for halogenation.

The methylerythritol phosphate pathway as an oxidative stress sense and response system.

The methylerythritol phosphate (MEP) pathway is responsible for biosynthesis of the precursors of isoprenoid compounds in eubacteria and plastids. It is a metabolic alternative to the well-known mevalonate pathway for isoprenoid production found in archaea and eukaryotes. Recently, a role for the MEP pathway in oxidative stress detection, signalling, and response has been identified. This role is executed in part through the unusual cyclic intermediate, methylerythritol cyclodiphosphate (MEcDP). We postulate that this response is triggered through the oxygen sensitivity of the MEP pathway's terminal iron-sulfur (Fe-S) cluster enzymes. MEcDP is the substrate of IspG, the first Fe-S cluster enzyme in the pathway; it accumulates under oxidative stress conditions and acts as a signalling molecule. It may also act as an antioxidant. Furthermore, evidence is emerging for a broader and highly nuanced role of the MEP pathway in oxidative stress responses, implemented through a complex system of differential regulation and sensitivity at numerous nodes in the pathway. Here, we explore the evidence for such a role (including the contribution of the Fe-S cluster enzymes and different pathway metabolites, especially MEcDP), the evolutionary implications, and the many questions remaining about the behaviour of the MEP pathway in the presence of oxidative stress.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Molecular control of sucrose utilization in Escherichia coli W, an efficient sucrose-utilizing strain.

Sucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization in Escherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes in E. coli W were examined by knockout and overexpression experiments. At low sucrose concentrations, the csc genes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout of cscR and cscK conferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism in E. coli W, demonstrating that no other genes can provide sucrose transport or inversion activities. However, cscK is not essential for sucrose utilization. Fructose is excreted into the medium by the cscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression of cscA, cscAK, or cscAB could complement the WΔcscRKAB knockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressing cscAB, and full growth rate complementation in WΔcscRKAB also required cscAB. Our understanding of sucrose utilization can be used to improve E. coli W and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.