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StartBiotechnologyAn in vivo gene amplification system for prime degree expression in Saccharomyces...

An in vivo gene amplification system for prime degree expression in Saccharomyces cerevisiae


Assemble design for in vivo gene amplification

Two parts are required for gene amplification to happen: (1) a gene linked to cell health, and (2) homologous DNA sequences to help recombination20. As well as, a powerful replication origin can promote amplification29,30,31. These three parts exist in tandem repeat within the rDNA area and the CUP1 area within the yeast genome (Fig. 1a).

Fig. 1: Design of in vivo gene amplification.
figure 1

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

We designed a genetic construction for gene amplification in yeast (Fig. 1b). The assemble has recombination arms at every finish. Arm 1 is homologous to the promoter area of a haploinsufficient gene, and Arm 2 is homologous to the preliminary a part of the haploinsufficient gene open studying body. This enables insertion of the assemble into the genome by homologous recombination. Downstream of Arm 1 are a selectable marker for transformation choice and homologous Arm 3, which is homologous to the terminator area of the haploinsufficient gene. Between Arm 3 and Arm 2, there are an autonomous replicating sequence (ARS) and a promoter. The promoter is weaker than the native promoter of the haploinsufficient gene and positioned such that integration leads to substitution of the native promoter of the haploinsufficient gene with the weaker promoter. Genes of curiosity, to be expressed heterologously, might be inserted between Arm 3 and the weaker promoter.

Driving expression by way of a weaker promoter attenuates the protein yield from every copy of the haploinsufficient gene. This, in flip, is predicted to lower the expansion price in yeast. Native amplification of the area between homologous Arm 3 will then happen as yeast evolves in the direction of sooner progress.

Utilizing RPL25 or SEC23 haploinsufficient gene loci to drive amplification

The impact of haploinsufficient genes on progress health has been characterised beforehand28. We used the ribosomal 60S subunit protein L25 (RPL25) and the SEC23-encoding part of the Sec23p-Sec24p heterodimer of the COPII vesicle coat. These two genes have the strongest health impact in wealthy medium and in minimal mineral medium28. We developed 4 constructs with RPL25 because the driving gene, LEU2 as choice marker, and an early-firing ARS ARS30632 to facilitate amplification; and three constructs with SEC23 because the driving gene, hygromycin B resistant gene hphMX as choice marker, and the robust ARS1max ARS33 to facilitate amplification (Fig. 2a).

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

a Schematic of gene amplification constructs. b, c, e Most progress price, yEGFP (yeast-enhanced inexperienced fluorescent protein) gene copy quantity, and yEGFP fluorescence in strains reworked with the constructs in a. Strains had been chosen by brightness of yEGFP fluorescence (Supplementary Fig. 1). d Promoter characterisation utilizing yEGFP because the reporter within the cells on the exponential progress part (EXP) and the post-diauxic-shift progress part (ETH) when ethanol was used because the carbon supply. Yeast cells had been grown in microplates in d and in flasks in b, c, e. yEGFP fluorescence is expressed as proportion of exponential-phase auto-fluorescence of the reference pressure. The numbers had been calculated by dividing the imply worth for RPL25 or SEC23 (underlined) by the imply worth. Imply values ± customary deviations are proven (N = 3 unbiased organic replicates). Supply information are offered as a Supply Knowledge file.

To determine promoters with appropriate expression strengths, promoters had been chosen from the big variety of promoters we beforehand analysed34, to check with every goal locus (Fig. 2a, d). For the RPL25 constructs we used the YEF3 promoter (which has comparable energy to the RPL25 promoter; Assemble 1) and the ERG1, PDA1, or BTS1 promoters (all with multiple-fold weaker expression than RPL25 promoter; Constructs 2–4). For the SEC23 constructs, we used the ERG1 promoter (stronger than the SEC23 promoter; Assemble 5), the GLO2 promoter, or the COG7 promoter (each multiple-fold weaker than the SEC23 promoter; Constructs 6 and seven). An eighth promoter assemble was designed and examined later (see beneath). We used yeast-enhanced inexperienced fluorescent protein (yEGFP) underneath the management of the TEF1 promoter and the URA3 terminator because the gene of curiosity and as a reporter for proof of idea.

The seven constructs had been reworked into S. cerevisiae CEN.PK strains. Transformation plates had been screened by imaging yEGFP fluorescence underneath blue gentle (Supplementary Fig. 1a, c) and colonies had been chosen for elevated fluorescence. For every assemble, six strongly fluorescing clones had been chosen. Visible statement after sub-culturing demonstrated an inverse correlation between promoter energy (Fig. second) and GFP fluorescence (Supplementary Fig. 1b). Three clones with comparable fluorescence had been chosen for quantitative characterisation for every assemble.

The place promoter energy was comparable or larger than the native promoter, yEGFP was discovered at a single copy on the genome (Fig. 2c: Constructs 1 and 5), and fluorescence (Fig. 2e: Constructs 1 and 5) was much like fluorescence we noticed beforehand in strains with a single copy of the PTEF1-yEGFP-TURA3 assemble3. yEGFP gene copy quantity and fluorescence each elevated the place the native promoter was substituted for weaker promoters (Fig. 2c, e: Constructs 2–4, 6, 7). Copy quantity elevated from 4-fold to 47-fold, whereas fluorescence improve was 4-fold to 92-fold. There was a powerful optimistic correlation between copy quantity and fluorescence (r2 = 0.985), and a weak destructive correlation between fluorescence and promoter energy/copy quantity (r2 = 0.376 and 0.694 respectively). Essentially the most exceptional end result was the place the RPL25 promoter was substituted for the BTS1 promoter; this resulted in ~47 copies of yEGFP per genome and a ~92-fold improve yEGFP fluorescence (Fig. 2c, e).

To additional improve copy quantity on the SEC23 locus, we attenuated translation by making a assemble with three non-preferred glycerine codons (GGA) inserted following the beginning codon of SEC23 underneath the management of the COG7 promoter (Fig. 2a: Assemble 8), which delivered probably the most gene amplification within the first spherical (9 copies). A slight improve in gene copy and fluorescence was obtained (Fig. 2c, e). Translational downregulation by use of non-preferred codons offers a second mechanism to drive a rise in copy quantity for genes at haploinsufficient gene loci.

Within the preliminary design (Fig. 1), we embrace ARS within the module basing on the genetic options at naturally amplified genomic loci. To substantiate the function of ARS within the present system, we eliminated the ARS sequence within the Assemble 3. The ARS-removed assemble may result in the formation of the very fluorescent colonies after transformation (Supplementary Fig. 1). This means that ARS is probably not important for HapAmp.

Elevated copy quantity didn’t negatively affect the expansion price of any of the strains aside from clones with the PBTS1-RPL25 assemble (Fig. 2b), which had an exceptionally excessive integration copy quantity (Fig. 2c). This pressure confirmed an ~7% lower in progress price (two-tailed t-test p = 0.001).

Lengthy-read sequencing on strains containing Constructs 3 and 4 confirmed that the constructs had been built-in into the RPL25 (YOL127W) locus and that yEGFP-RPL25 sequences had been amplified in tandem repeat constructions (Supplementary Figs. 2 and 35). The pressure expressed the very best degree of yEGFP (Assemble 4) was sub-cultured in yeast extract-peptone-glucose medium for ~48 generations for stability check (Supplementary Fig. 6). GFP fluorescence ranges and inhabitants homogeneity didn’t change, indicating that HapAmp is genetically secure.

Enhancing heterologous manufacturing of the sesquiterpene trans-nerolidol

We examined the efficiency of the HapAmp methodology utilizing sesquiterpene (C15; trans-nerolidol) manufacturing. We used a background pressure with an upregulated mevalonate pathway for manufacturing of terpene precursors (o401R)35,36,37,38. On this pressure, the GAL80 repressor gene is disrupted permitting diauxic induction of GAL promoters, that are used to manage transgenes.

We constructed a reference pressure N401-1 harbouring a multi-copy 2μ plasmid pJT9RFR39 (Fig. 3a) with overexpression cassettes for farnesyl pyrophosphate synthase (ERG20) and nerolidol synthase (Ac.NES1). The nerolidol synthase cassette features a fluorescence-activating and absorption-shifting tag (Y-FAST)40 and a 2A peptide from Equine rhinitis B virus 141 fused to the N-terminus of nerolidol synthase. This enables Y-FAST fluorescence for use as a proxy for nerolidol synthase expression39.

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

a, b Schematic map of genetic vectors used to introduce nerolidol artificial genes into yeast. ch Pressure 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 closing focus 20 μM was added to the yeast samples earlier than movement cytometry assay, and is expressed as fold-change of exponential-phase auto-fluorescence of the reference pressure GH43. Nerolidol manufacturing at 72 h was proven. Kernel density was calculated with bandwidth equal to 0.05. Imply values ± customary deviations are proven (cf, h; N = 4 unbiased organic replicates). Two-tailed Welch’s t-test was used for evaluating two teams, and p values had been proven in d, h. Supply information are offered as a Supply Knowledge file.

The nerolidol synthase expression cassette (Y-FAST-2A-Ac.NES1) was cloned into the RPL25 insertion vector within the amplification area with three completely different promoters for substitute of the RPL25 promoter; the ERG20 expression cassette was cloned on the non-amplification area (Fig. 3b). Colonies with vibrant Y-FAST fluorescence had been chosen from the transformation plates. This delivered strains N401-2, N401-3, & N401-4 (promoters PERG1, PPDA1, and PBTS1, respectively).

In comparison with the reference pressure N401-1, these three strains exhibited sooner progress (Fig. 3c, d), larger Y-FAST fluorescence (Fig. 3f), and better nerolidol manufacturing (Fig. 3h). The Y-FAST-2A-Ac.NES1 cassette was efficiently amplified in vivo within the three check strains (Fig. 3e).

The reference 2μ plasmid pressure harboured 14 copies of the Y-FAST-2A-AcNES1 assemble, much like pressure N401-3, and better than that in pressure N401-2. Nevertheless, N401-1 had the bottom Y-FAST fluorescence (Fig. 3f). The discrepancy between copy quantity and fluorescence was attributable to lack of induction of Y-FAST expression in a big proportion of N401-1 cells (Fig. 3g). In distinction to the 2μ plasmid pressure, the strains harbouring the in vivo amplification constructs confirmed higher synchronicity for Y-FAST induction (Fig. 3g N401-3; others not proven). This may increasingly contribute to the improved manufacturing.

Enhancing heterologous manufacturing of the monoterpene limonene

We subsequent examined the system on manufacturing of monoterpenes (C10). Monoterpene manufacturing requires introduction of a devoted C10 geranyl pyrophosphate (GPP) synthase42. We’ve beforehand used an Erg20pN127W mutant42, which excludes the C15 chain from the lively website to generate a GPP pool, together with focused degradation of the endogenous C15 synthase Erg20p by way of protein degron tags35,39 to lower competitors on the C10 node by Erg20p and redirect GPP in the direction of monoterpene manufacturing. In mevalonate pathway-enhanced strains, this strategy delivered lower than 100 mg l−1 monoterpene—an order of magnitude beneath the degrees achieved for sesquiterpene engineering.

We used a mevalonate pathway-enhanced pressure with the endogenous Erg20p underneath an auxin-inducible protein degradation mechanism39 as a background pressure to minimise flux competitors by way of the native sterol pathway. Two completely different promoter constructs had been developed for amplification of the limonene artificial module (Fig. 4a). The amplified area contained a fusion of a number of genes: Y-FAST-2A39, the maltose-binding protein from E. coli for improved solubility43, a brief linker, limonene synthase from Citrus limon35, a 6*glycerine linker, and the Erg20p N127W F96W mutant42 (which has a better particular GPP manufacturing price than the Erg20pN127W mutant) as a GPP synthase. This fusion assemble was underneath the management of the GAL2 promoter from S. kudriavzevii44. The 2 constructs had been reworked into the RPL25 locus within the background pressure, delivering strains LIM141M (PPDA1) and LIM141MH (PBTS1).

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

LIM141R2 is certainly one of LIM141R organic replicates. a Schematic map of genetic vectors used to introduce limonene artificial genes into yeast. bf Pressure characterisation in two-phase flask cultivation with 20 g l−1 glucose and dodecane overlay. Artificial auxin 1-Naphthaleneacetic acid (NAA) was added to 1 mM on the late exponential progress part (OD > 4). Y-FAST fluorescence was measured after 4-hydroxy-3-methylbenzylidene rhodanine (HMBR) with closing focus 20 μM was added to the yeast samples earlier than movement cytometry assay and is expressed as fold-change of exponential-phase auto-fluorescence of the reference pressure GH430. Limonene and geraniol manufacturing at 96 h was proven. Imply values ± customary deviations are proven (N = 3 unbiased organic replicates for LIM141R, LIM141M and three unbiased cultures for LIM141R2 in bf. N = 4 unbiased organic replicates in be and three unbiased organic replicates in f for LIM141MH). Supply information are offered as a Supply Knowledge file.

For the reference pressure, the assemble was launched into the background pressure by way of a 2μ plasmid (Fig. 4a). We characterised 4 organic replicates (LIM141R representing three organic replicates and LIM141R2 representing one organic replicate; Fig. 4). On this case, 2μ plasmid delivered ~2 copies per genome of the limonene synthase/Y-FAST module (proven by Y-FAST copy quantity; Fig. 4c). LIM141R, the three organic replicates produced ~40 mg l−1 limonene (Fig. 4f), the titre similar to a earlier pressure LIM141 expressing limonene synthase and Erg20pN127W with out gene fusion39. Nevertheless, one organic replicate (LIM141R2, Fig. 4) produced ~300 mg l−1 limonene. LIM141R2 exhibited sooner progress and better Y-FAST fluorescence ranges than different three organic replicates (LIM141R, Fig. 4b, d, e). The development in LIM141R2 could also be brought on by unintended genetic variations.

Harbouring HapAmp limonene artificial module, each strains LIM141M and LIM141MH produced an order of magnitude extra limonene than LIM141R and former efforts utilizing 2µ plasmids35,39, with the perfect manufacturing, ~0.95 g l−1 limonene at 96 h, by pressure LIM141M (Fig. 4f). This titre is 5.6-fold larger than the earlier highest titre ever obtained in yeast45, and ~2-fold larger than the perfect titres achieved in batch cultivation in E. coli46,47. Pressure LIM141MH confirmed a slower exponential progress and the decrease ranges of Y-FAST fluorescence in comparison with pressure LIM141M (Fig. 4b, d, e), regardless of having extra copies of the limonene synthase/Y-FAST module (proven by Y-FAST copy quantity; Fig. 4c). Each strains additionally amassed ~12 mg l−1 of the monoterpene alcohol geraniol, which is usually produced by yeast with an elevated GPP pool35,39. No farnesol (C15 alcohol) or geranylgeraniol (C20 alcohol) had been amassed by the strains, indicating that subcellular swimming pools of FPP and the C20 geranylgeranyl pyrophosphate (GGPP) had been low, and that amplification of limonene artificial module led to vital redirection of the carbon flux in the direction of monoterpene manufacturing.

Enhancing heterologous tetraterpenoid lycopene manufacturing in yeast

A 3-gene lycopene artificial module managed by GAL promoters was beforehand constructed in a 2μ plasmid37 (Fig. 5a). This assemble consists of the farnesyl pyrophophase mutant gene ERG20F96C which produces GGPP48, a phytoene synthase49,50, and a lycopene-forming phytoene desaturase mutant50. This plasmid was reworked right into a mevalonate pathway-enhanced background pressure, producing pressure LYC137. This pressure amassed ~5 mg lycopene per gram of biomass in 120-h flask cultivation (Fig. 5b).

Fig. 5: Characterisation of lycopene-producing strains with lycopene artificial genes built-in at amplified RPL25 locus.
figure 5

a Schematic maps of genetic vectors used to introduce lycopene artificial genes into yeast. b Lycopene manufacturing in flask cultivations. Yeast cells in exponential progress was inoculated into 20 ml MES-buffered YNB medium with 20 g l−1 glucose in 125 ml Erlenmeyer flask to begin a tradition at OD600 = 0.2. Imply values ± customary deviations are proven (N = 4 unbiased organic replicates). Supply information are offered as a Supply Knowledge file.

The lycopene artificial module was sub-cloned into each the PDA1 and BTS1 promoter RPL25-driving HapAmp vectors (Fig. 5a). The ensuing constructs had been reworked into the identical background pressure, producing strains LYC4 and LYC5, respectively. Pressure LYC4 (PPDA1-RPL25) amassed barely extra lycopene than pressure LYC1, though the rise was not vital (Fig. 5b). Pressure LYC5 amassed ~25 mg lycopene per gram of biomass, five-fold larger than pressure LYC1 (Fig. 5b).

Excessive-level expression of heterologous proteins in yeast

S. cerevisiae can be utilized as a platform organism for protein manufacturing, together with manufacturing of pharmaceutical proteins. Nevertheless, a infamous drawback is that heterologous proteins manufacturing just isn’t as excessive as what’s achievable with E. coli expression techniques. The high-level expression in E. coli might be attributed to the utilization of high-copy-number plasmids (such because the widespread pET vectors with copy quantity about ~15–20) and using a really robust inducible promoter51. We used the PBTS1-RPL25-driving HapAmp constructs to introduce the AeBlue chromoprotein gene52 (Fig. 6a) or the EforRed chromoprotein gene53. Blue or pink colonies had been obtained on the transformation plates (Supplementary Fig. 7), indicating high-level expression of the chromoproteins.

Fig. 6: Characterisation of the expression of heterologous proteins (AeBlue and HPV16 capsid L1) by way of multi-copy genome integration (MI) utilizing PBTS1-RPL25-driven in vivo gene amplification.
figure 6

a Schematic of genetic vectors used to precise AeBlue and HPV16 L1. b cells harbouring an empty 2μ, the amplifiable AeBlue assemble (MI), AeBlue-and-HPV16-L1 2μ plasmid, and amplifiable AeBlue-and-HPV16-L1 assemble (MI). Cells had been grown in MES-buffered YNB medium with 20 g l−1 glucose and picked up at 72 h, or had been 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 (proven 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 pattern from c. Experimental repetition just isn’t accomplished for c and d. Numbers in bd are for pattern cross-reference. The bands d1, d2, d3, and d4 are analysed utilizing a LC-MS/MS-based proteomic methodology (Supplementary Methodology 1), and the information can be found in Supplementary Knowledge 1 (d1), Supplementary Knowledge 2 (d2), Supplementary Knowledge 3 (d3), and Supplementary Knowledge 4 (d4). Supply information for VLPs in d are offered as a Supply Knowledge File.

Having confirmed that the chromoproteins had been efficient markers, we then inserted a human papillomavirus (HPV) 16 main capsid protein L1 gene after the AeBlue expression cassette (Fig. 6a) to check the system for manufacturing of a pharmaceutical protein. For a reference, we cloned AeBlue-and-HPV16-L1 expression cassettes right into a yeast 2μ plasmid (Fig. 6a). To match the effectivity of protein manufacturing in numerous techniques, an empty 2μ plasmid, the AeBlue-and-HPV16-L1 2μ plasmid, the RPL25-amplifiable AeBlue assemble, and the RPL25-amplifiable AeBlue-and-HPV16-L1 assemble had been reworked individually into CEN.PK (gal80Δ). The 4 ensuing strains had been grown in MES-buffered YNB medium with 20 g l−1 glucose aerobically for 72 h. Cells with multi-copy integration of the AeBlue expression cassette confirmed a powerful Tibetan blue color, whereas cells with an empty cassette had been milky white color (Fig. 6b). The cells with 2μ plasmid containing AeBlue + HPV-L1 expression cassettes had been a faint blue color, whereas the cells with multi-copy integration of AeBlue + HPV-L1 expression cassettes displayed the robust Tibetan blue color (Fig. 6b). This indicated superior expression capability from the in vivo amplification methodology for multi-copy genome integration, in comparison with standard 2μ plasmid methodology.

SDS-PAGE of whole-cell and soluble protein extracts confirmed bands at ~25 kD (AeBlue molecular weight) in all samples, with a lot stronger bands noticed within the multi-copy integration pressure samples than within the 2μ plasmid pressure samples (Fig. 6d). Within the multi-copy integration strains, these bands represented ~3% of whole-cell protein, suggesting heterologous protein expression in yeast might attain the degrees usually obtained in E. coli.

A second robust band at ~50 kD band (HPV16-L1 molecular weight) was noticed in samples from cells expressing HPV-L1, though it was not as distinct on the putative AeBlue band (Fig. 6d). This can be attributable to using the Se.GAL2 promoter, which isn’t totally induced within the ethanol part, in these constructs in comparison with the constitutive ALD6 promoter used for the AeBlue expression cassette. Once more, the bands within the multi-copy integration pressure samples had been stronger than the 2μ plasmid samples. Surprisingly, contemplating that HPV16-L1 is a soluble protein54, these bands weren’t distinguishable in lysate supernatant samples.

To completely induce the Se.GAL2 promoter for HPV16-L1 expression, we tried to develop the plasmid and integration strains harbouring HPV16-L1 in artificial minimal medium (YNB) with ethanol or galactose because the carbon supply. Nevertheless, these cultivation circumstances had been deadly for the multi-copy-integration cells. We then grew the cells in wealthy (yeast-peptone (YP)) medium with 20 g l−1 galactose because the carbon supply. Beneath these circumstances, AeBlue expression from 2μ plasmid was not observable by visible examination (Fig. 6b) or SDS-PAGE (Fig. 6d). This can be attributable to lack of 2μ plasmid within the wealthy medium. In distinction, robust AeBlue-specific and HPV16-L1-specific bands had been seen in whole-cell lysate and lysate supernatant samples from the cells with multi-copy integration constructs. This additional confirmed that HPV16 L1 capsid protein is insoluble in yeast in our system. Makes an attempt to solubilise HPV16-L1 L1 capsid protein had been unsuccessful (information not proven). Regardless of being unable to detect HPV16-L1-specific bands in lysate supernatant (Fig. 6d), we may nonetheless separate correctly assembled virus-like particles (VLPs) by ultracentrifugation of lysate supernatant (Fig. 6c). SDS-PAGE examination of VLP parts purified from ultracentrifugation confirmed a HPV16-L1-specific band at ~50 kD (Fig. 6d; Lane VLPs:4). TEM photos of the VLPs confirmed their diameter was round 40 nm (Fig. 6c), in keeping with earlier literature55.

In SDS-PAGE outcomes, we noticed robust bands within the lysate supernatant pattern (band d1) and lysate pellet samples (bands d2, d3, and d4) (Fig. 6d). LC-MS/MS-based proteomics was used to analyse the protein composition in these 4 bands (Supplementary Knowledge 14). The highest hit protein within the ~50 kD band (band d2) was the HPV16 L1 capsid. Curiously, the highest hit proteins in different three bands (d1, d3 and d4) had been yeast chaperones. In bands d1 and d3, the highest hit proteins had been HSP70 household chaperone Ssa1, and in bands d4, the highest hit protein was HSP90 household chaperone Hsc82. We due to this fact hypothesised that insoluble expression of HPV16-L1 induced upregulation of yeast chaperones, and HPV16-L1, HSP70 chaperones, and HSP90 chaperones would possibly exist in insoluble types. Nevertheless, it will require additional systematic examination to get a greater understanding of those phenomena.

In abstract, though some insoluble expression of the HPV16 L1 was noticed, our outcomes each with chromoprotein AeBlue and the HPV16 L1 confirmed that multi-copy gene integration by way of HapAmp methodology can result in heterologous protein overexpression in yeast to the excessive ranges which might be generally seen in E. coli expression techniques.

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