An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae

Abstract

Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L^-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.

Fig. 1. Design of in vivo gene amplification.

a Natural genome structures at the ribosomal DNA (rDNA) locus on chromosome XII and the CUP1 locus on chromosome VII. b Construct design for in vivo gene amplification (HapAmp). ARS autonomous replicating sequence.

Fig. 2. Design and characterisation of gene amplification constructs for haploinsufficient target genes RPL25 or SEC23.

a Schematic of gene amplification constructs. b, c, e Maximum growth rate, yEGFP (yeast-enhanced green fluorescent protein) gene copy number, and yEGFP fluorescence in strains transformed with the constructs in a. Strains were selected by brightness of yEGFP fluorescence (Supplementary Fig. 1). d Promoter characterisation using yEGFP as the reporter in the cells at the exponential growth phase (EXP) and the post-diauxic-shift growth phase (ETH) when ethanol was used as the carbon source. Yeast cells were grown in microplates in d and in flasks in b, c, e. yEGFP fluorescence is expressed as percentage of exponential-phase auto-fluorescence of the reference strain. The numbers were calculated by dividing the mean value for RPL25 or SEC23 (underlined) by the mean value. Mean values ± standard deviations are shown (N = 3 independent biological replicates). Source data are provided as a Source Data file.

Fig. 3. Characterisation of nerolidol-producing strains, harbouring nerolidol synthetic genes on a 2μ plasmid (N401-1) or integrated at amplified RPL25 locus (N401-2, N401-3, and N401-4).

a, b Schematic map of genetic vectors used to introduce nerolidol synthetic genes into yeast. c–h Strain characterisation in two-phase flask cultivation with 20 g l⁻¹ glucose and dodecane overlay. Y-FAST (fluorescence-activating and absorption-shifting tag) fluorescence was measured after 4-hydroxy-3-methylbenzylidene rhodanine (HMBR) with final concentration 20 μM was added to the yeast samples before flow cytometry assay, and is expressed as fold-change of exponential-phase auto-fluorescence of the reference strain GH4. Nerolidol production at 72 h was shown. Kernel density was calculated with bandwidth equal to 0.05. Mean values ± standard deviations are shown (c–f, h; N = 4 independent biological replicates). Two-tailed Welch’s t-test was used for comparing two groups, and p values were shown in d, h. Source data are provided as a Source Data file.

Fig. 4. Characterisation of limonene-producing strains with limonene synthetic genes on a 2μ plasmid (LIM141R and LIM141R2) or integrated at amplified RPL25 locus (LIM141M and LIM141MH).

LIM141R2 is one of LIM141R biological replicates. a Schematic map of genetic vectors used to introduce limonene synthetic genes into yeast. b–f Strain characterisation in two-phase flask cultivation with 20 g l⁻¹ glucose and dodecane overlay. Synthetic auxin 1-Naphthaleneacetic acid (NAA) was added to 1 mM at the late exponential growth phase (OD > 4). Y-FAST fluorescence was measured after 4-hydroxy-3-methylbenzylidene rhodanine (HMBR) with final concentration 20 μM was added to the yeast samples before flow cytometry assay and is expressed as fold-change of exponential-phase auto-fluorescence of the reference strain GH4. Limonene and geraniol production at 96 h was shown. Mean values ± standard deviations are shown (N = 3 independent biological replicates for LIM141R, LIM141M and three independent cultures for LIM141R2 in b–f. N = 4 independent biological replicates in b–e and three independent biological replicates in f for LIM141MH). Source data are provided as a Source Data file.

Fig. 5. Characterisation of lycopene-producing strains with lycopene synthetic genes integrated at amplified RPL25 locus.

a Schematic maps of genetic vectors used to introduce lycopene synthetic genes into yeast. b Lycopene production in flask cultivations. Yeast cells in exponential growth was inoculated into 20 ml MES-buffered YNB medium with 20 g l⁻¹ glucose in 125 ml Erlenmeyer flask to start a culture at OD₆₀₀ = 0.2. Mean values ± standard deviations are shown (N = 4 independent biological replicates). Source data are provided as a Source Data file.

Fig. 6. Characterisation of the expression of heterologous proteins (AeBlue and HPV16 capsid L1) via multi-copy genome integration (MI) using P_BTS1-RPL25-driven in vivo gene amplification.

a Schematic of genetic vectors used to express AeBlue and HPV16 L1. b cells harbouring an empty 2μ, the amplifiable AeBlue construct (MI), AeBlue-and-HPV16-L1 2μ plasmid, and amplifiable AeBlue-and-HPV16-L1 construct (MI). Cells were grown in MES-buffered YNB medium with 20 g l⁻¹ glucose and collected at 72 h, or were grown in YP medium with 20 g l⁻¹ galactose to OD₆₀₀ = ∼20. c Ultracentrifugation of the supernatant on an iodixanol gradient to separate a band containing HPV16-L1 virus-like particles (shown by orange arrow), and transmission electron microscopy confirming the presence of HPV16-L1 virus-like particles (VLPs). d SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) for whole-cell lysates, lysate supernatant, and lysate pellets of yeast samples in b, and VLPs sample from c. Experimental repetition is not done for c and d. Numbers in b–d are for sample cross-reference. The bands d1, d2, d3, and d4 are analysed using a LC-MS/MS-based proteomic method (Supplementary Method 1), and the data are available in Supplementary Data 1 (d1), Supplementary Data 2 (d2), Supplementary Data 3 (d3), and Supplementary Data 4 (d4). Source data for VLPs in d are provided as a Source Data File.