MOESM1 of Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast

Additional file 1: Table S1. Primers and PCR fragments amplified/used in this work. PXXX, promoter of gene XXX; T XXX , terminator of gene XXX; Y-GDNA, CEN.PK113-7D genomic DNA; sequence annealing to template in primers is shown in red and italics; over-lap sequence for over-lap extension PCR and Gibson Assembly is underlined; restriction sites used in cloning are shown in bold. Table S2. Molecular construction of plasmids used in this work. Figure S1. Plasmid rearrangement in the strain NC1D. Plasmid pPMVAd36 and [TRP1] from NC1D were digested by restriction enzymes NotI, SalI, SphI, BamHI and SbfI and gel figure was shown as the right-bottom figure. Figure S2. The growth profile of strain GH4 [CEN.PK113-5D derivative; ura3(1, 704 )::KlURA3] (1) on sucrose. The cells were pre-cultured on 40 g L-1 glucose. Mean values from duplicate experiments are shown. Figure S3. Logic charts for fed-batch feeding scripts: (a) carbon-source-restricted/DO-triggered fed-batch cultivation; (b) carbon-source-overflowed/carbon-source-pulsing fed-batch cultivation. Fs, feeding flow storage value; DOt, dissolved oxygen on-line value at time t; DOL, lowest dissolved oxygen storage value; T1, storage time; t, on-line time; Îź, specific rate of feeding flow increasement; N, agitatation speed; Nmax, the maxium agitation speed; FVs, feeding volume storage value; FVt, feeding volume on-line value; Vt, culture volume on-line value. Figure S4. Growth (OD600) and process values (Dissovled oxygen, DO; oxygen transfer rate, OTR; carbon transfer rate; CTR; respiration quotient, RQ) in fed-batch cultivation for strain N391DA, with feeding logics in Fig. S1a employed. (a&b), 600 g L-1 glucose feeding; (c&d), 600 g L-1 sucrose feeding; (e&f), 400 g L-1 glucose and 158 g L-1 ethanol feeding. Figure S5. Growth (OD600) and process values (Dissovled oxygen, DO; oxygen transfer rate, OTR; carbon transfer rate; CTR; respiration quotient, RQ) in fed-batch cultivation for strain N391DA, with feeding logics in Fig. S1b employed. (a&b), 600 g L-1 glucose feeding with 10 g L-1 glucose pulse; (c&d), 600 g L-1 glucose feeding with 20 g L-1 glucose pulse; (e&f), 600 g L-1 sucrose feeding with 20 g L-1 sucrose pulse. Figure S6. The influence of nerolidol on yeast growth. Synthetic minimal medium was used, which contained 6.7 g L-1 yeast nitrogen base (Sigma-Aldrich #Y0626; pH 6.0) and 20 g L-1 glucose. Isomer-mixed nerolidol (Sigma-Aldrich #H59605) was used. Tween 80 was added to homogenize nerolidol into liquid medium. Mean values Âą standard deviations are shown (N = 3)

An Expanded Heterologous <i>GAL</i> Promoter Collection for Diauxie-Inducible Expression in <i>Saccharomyces cerevisiae</i>

The <i>GAL</i> promoters are applied in metabolic engineering and synthetic biology to control gene expression in the budding yeast <i>Saccharomyces cerevisiae</i>. In <i>gal80Δ</i> background strains, they show diauxie-inducible expression, a feature beneficial in metabolic pathway optimization. However, only a limited number of <i>GAL</i> promoters have been characterized and are available for engineering purposes. Multiple uses of the same promoters can result in genetic instability in engineered strains due to homologous recombination. Here, 11 <i>GAL1</i>/2 promoters from other <i>Saccharomyces</i> species were isolated and characterized in <i>S. cerevisiae</i>. They exhibited diauxie-inducible expression patterns with low strength in exponential growth phase and induction in the ethanol growth phase. These promoters represent an expansion to the collection of <i>GAL</i> promoters available for genetic engineering in <i>S. cerevisiae</i>, including an increased diversity of expression levels. This provides the capacity for increased numbers of genetic manipulations with a lower risk of genetic instability

Additional file 1: of Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities

Table S1. Fluorescence of the destabilized GFP (yEGFP-CLN2 PEST) in the strains cultivated on 2% v/v ethanol to OD600 = 0.90 ± 0.03. Table S2. Effect of difference glucose concentrations (linear regression) and different carbon sources (one-way ANOVA) on GFP fluorescence driven by various promoters. Table S3. The primers, the plasmids and the strains used in this work. Figure S1. Pre-evaluation of mid-log phase for microplate cultivation. Figure S2. Correlation of GFP fluorescence determination in the strains using either the destabilized GFP (yEGFP-CLN2 PEST) or the normal GFP (yEGFP) as the reporter. Figure S3. Post-hoc test for fluorescence levels (sorted from low to high) of various promoter-yEGFP strains on different carbon source. Figure S4. Yeast cultures with/without copper addition. Figure S5. De-repression of ADH2 promoter

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

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 causethe 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

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

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 causethe 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

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

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 causethe 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