Avoiding overflow metabolite formation in Komagataella phaffii fermentations to enhance recombinant protein production.

BACKGROUND: Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. RESULTS: High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. CONCLUSIONS: These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii.

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production.

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (PG1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. PG1 and its improved derivatives PG1-3, D-PGS4 and D-PGS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, PG1-3-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the PG1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

Optimal fermentation conditions for growth and recombinant protein production in Pichia pastoris: Strain selection, ploidy level and carbon source.

High-cell-density fermentation is a critical aspect of industrial protein production, requiring the selection of an optimal growth medium and carbon source. Pichia pastoris, a methylotrophic yeast, has been established as a widespread recombinant protein expression system in the food and pharmaceutical industries. The primary objective of this work was to create a superior platform for producing alternative proteins thus contributing to future innovation in these sectors. This study compared three wild-type strains, with two of them also analyzed in their diploid versions, using shake flasks and bioreactors. It investigated glucose and glycerol as carbon sources using mCherry as a protein model. Glycerol emerged as the preferred carbon source, resulting in over 40% increase in biomass concentrations compared to glucose across all strains. Notably, wild-type strain Y-7556 reached an exceptional biomass concentration of 244 g DCW/L in just 48 h, the highest reported to date, highlighting the potential of high-cell-density fermentation in P. pastoris. Regarding protein expression, the diploid version of Y-11430 produced >43% of purified mCherry protein after 123 h of fermentation, compared to the haploid counterpart. Our findings underscore the advantages of diploid strains, optimized fermentation media, and carbon source selection, effectively addressing crucial gaps in the literature.

Heterologous expression, characterisation and 3D-structural insights of GH18 chitinases derived from sago palm (Metroxylon sagu).

Although plants don't have chitins, they produce chitinases to protect themselves from biotic and abiotic stressors. There are two forms of chitinases found in organisms: glycosyl hydrolase 18 (GH18) and 19 (GH19) families. Plant GH19 chitinases are well known for their role in protecting against pathogens, but the roles of GH18 chitinases have not been fully elucidated. This study aimed to produce and characterise two recombinant GH18 chitinases from Metroxylon sagu. Two GH18 chitinase genes, MsChi1 and MsChi2, were identified, with nucleotide sequences of 1009 and 1308 bp, respectively. The proteins encoded by MsChi1 and MsChi2 genes were single polypeptide chains of 310 and 300 amino acids with predicted molecular masses of 31.21 and 30.15 kDa, respectively. Both cDNAs were cloned and expressed in the GS115 strain of Pichia pastoris. Recombinant MsChi1 and MsChi2 exhibited optimal activity at 60 °C with acidic pH 4.0 and 5.0, respectively. Both recombinant enzymes could hydrolyze synthetic and natural substrates (colloidal chitin). rMsChi1 preferred 4-nitrophenol N,N'-diacetyl-ß-D chitobioside, while rMsChi2 preferred 4-nitrophenol N,N',N″-triacetyl-ß-D chitotriose, suggesting they might function as exochitinase and endochitinase, respectively. They also demonstrated antifungal activities against tested fungi. Homology modeling indicated ASP and GLU as essential residues for proton donation and acceptance.

Single Cell Expression Systems for the Production of Recombinant Proteins for Immunodiagnosis and Immunoprophylaxis of Toxoplasmosis.

Toxoplasmosis represents a significant public health and veterinary concern due to its widespread distribution, zoonotic transmission, and potential for severe health impacts in susceptible individuals and animal populations. The ability to design and produce recombinant proteins with precise antigenic properties is fundamental, as they serve as tools for accurate disease detection and effective immunization strategies, contributing to improved healthcare outcomes and disease control. Most commonly, a prokaryotic expression system is employed for the production of both single antigens and multi-epitope chimeric proteins; however, the cloning strategies, bacterial strain, vector, and expression conditions vary. Moreover, literature reports show the use of alternative microbial systems such as yeast or Leishmania tarentolae. This review provides an overview of the methods and strategies employed for the production of recombinant Toxoplasma gondii antigenic proteins for the serological detection of T. gondii infection and vaccine development.

A self-inducible heterologous protein expression system in Komagataella phaffii (Pichia pastoris).

Komagataella phaffii (previously described as Pichia pastoris) is a yeast that produces high-level heterologous proteins with a wide range of applications in medicine and industry. The methanol-induced alcohol oxidase I promoter (PAOX1) is frequently used for protein expression in this yeast. However, limitations on the use of methanol have been observed in large-scale production, including its flammability, toxicity, and need for special handling. Here, we propose to develop a system using recombinant cells constitutively expressing pectinmethyl esterase for expression of two reporter proteins, GFP and azurin, under the control of PAOX1 using pectin in production medium. So, this system is coherent with yeast culture medium containing pectin and heterologous gene inserted downstream of PAOX1 can be successfully expressed without the addition of methanol. Therefore, this novel Self-inducibLe heterologous protein EXpression (SILEX) system, which does not require the addition of methanol, can be used for the production of any protein. It can also be adapted for large-scale production. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04039-x.

Functional characterization and structural basis of an efficient ochratoxin A-degrading amidohydrolase.

Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an appealing strategy, and the search for more efficient enzymes is a prerequisite for achieving this goal. Here, a novel amidohydrolase, termed PwADH, was demonstrated to exhibit 7.3-fold higher activity than that of the most efficient OTA-degrading ADH3 previously reported. Cryo-electron microscopy structure analysis indicated that additional hydrogen-bond interactions among OTA and the adjacent residue H163, the more compact substrate-binding pocket, and the wider entry to the substrate-access cavity might account for the more efficient OTA-degrading activity of PwADH compared with that of ADH3. We conducted a structure-guided rational design of PwADH and obtained an upgraded variant, G88D, whose OTA-degrading activity was elevated by 1.2-fold. In addition, PwADH and the upgraded G88D were successfully expressed in the industrial yeast Pichia pastoris, and their catalytic activities were compared to those of their counterparts produced in E. coli, revealing the feasibility of producing PwADH and its variants in industrial yeast strains. These results illustrate the structural basis of a novel, efficient OTA-degrading amidohydrolase and will be beneficial for the development of high-efficiency OTA-degrading approaches.

Characterization of MAP c21873-1 as a new counter-selectable marker for unmarked genetic modification of Pichia pastoris.

BACKGROUND: Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity. RESULTS: At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully. CONCLUSIONS: We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.

Engineering high production of fatty alcohols from methanol by constructing coordinated dual biosynthetic pathways.

Microbial cell factories provide an efficient approach for the green manufacturing of chemicals. However, the excessive use of sugars increases the potential risk of food crisis. Methanol, an abundant feedstock, holds promise in facilitating low-carbon production processes. However, the current methanol bioconversion is hindered by limited regulatory strategies and relatively low conversion efficiency. Here, a yeast biocatalyst was extensively engineered for efficient biosynthesis of fatty alcohols through reinforcement of precursor supply and methanol assimilation in Pichia pastoris. Furthermore, the dual cytoplasmic and peroxisomal biosynthetic pathways were constructed by mating and exhibited robust production of 5.6 g/L fatty alcohols by using methanol as the sole carbon source. This study provides a heterozygous diploid P. pastoris strain with dual cytoplasmic and peroxisomal biosynthetic pathways, which achieved the highest fatty alcohol production from one-carbon feedstocks to date.

Expression in Pichia pastoris of Thermostable Endo-1,4-ß-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Engineering a versatile yeast platform for sesquiterpene production from glucose or methanol.

Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO2 and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. Pichia pastoris (syn. Komagataella phaffii), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, P. pastoris is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in P. pastoris with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules MK and PMK greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of ß-farnesene from dual carbon source by replacing the α-bisabolene synthase with a ß-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in P. pastoris. Moreover, it paves the way for the development of P. pastoris as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in P. pastoris.

Evaluation of the potential food allergy risks of human lactoferrin expressed in Komagataella phaffii.

Introduction: Prior to the introduction of novel food ingredients into the food supply, safety risk assessments are required, and numerous prediction models have been developed and validated to evaluate safety. Methods: The allergenic risk potential of Helaina recombinant human lactoferrin (rhLF, Effera™), produced in Komagataella phaffii (K. phaffii) was assessed by literature search, bioinformatics sequence comparisons to known allergens, glycan allergenicity assessment, and a simulated pepsin digestion model. Results: The literature search identified no allergenic risk for Helaina rhLF, K. phaffii, or its glycans. Bioinformatics search strategies showed no significant risk for cross-reactivity or allergenicity between rhLF or the 36 residual host proteins and known human allergens. Helaina rhLF was also rapidly digested in simulated gastric fluid and its digestibility profile was comparable to human milk lactoferrin (hmLF), further demonstrating a low allergenic risk and similarity to the hmLF protein. Conclusion: Collectively, these results demonstrate a low allergenic risk potential of Helaina rhLF and do not indicate the need for further clinical testing or serum IgE binding to evaluate Helaina rhLF for risk of food allergy prior to introduction into the food supply.

Biosynthesis of a Functional Fragment of Human Collagen II in Pichia pastoris.

Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.

Advances in Flavonoid and Derivative Biosynthesis: Systematic Strategies for the Construction of Yeast Cell Factories.

Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.

Anti-Oxidative and Anti-Apoptotic Oligosaccharides from Pichia pastoris-Fermented Cress Polysaccharides Ameliorate Chromium-Induced Liver Toxicity.

Oxidative stress impairs the structure and function of the cell, leading to serious chronic diseases. Antioxidant-based therapeutic and nutritional interventions are usually employed for combating oxidative stress-related disorders, including apoptosis. Here, we investigated the hepatoprotective effect of oligosaccharides, produced through Pichia pastoris-mediated fermentation of water-soluble polysaccharides isolated from Lepidium sativum (cress) seed mucilage, on chromium(VI)-induced oxidative stress and apoptosis in mice. Gel permeation chromatography (GPC), using Bio-Gel P-10 column, of the oligosaccharides product of fermentation revealed that P. pastoris effectively fermented polysaccharides as no long chain polysaccharides were observed. At 200 µg/mL, fractions DF73, DF53, DF72, and DF62 exhibited DPPH radical scavenging activity of 92.22 ± 2.69%, 90.35 ± 0.43%, 88.83 ± 3.36%, and 88.83 ± 3.36%, respectively. The antioxidant potential of the fermentation product was further confirmed through in vitro H2O2 radical scavenging assay. Among the screened samples, the highest H2O2 radical scavenging activity was displayed by DF73, which stabilized the free radicals by 88.83 ± 0.38%, followed by DF53 (86.48 ± 0.83%), DF62 (85.21 ± 6.66%), DF72 (79.9 4± 1.21%), and EPP (77.76 ± 0.53%). The oligosaccharide treatment significantly alleviated chromium-induced liver damage, as evident from the increase in weight gain, improved liver functions, and reduced histopathological alterations in the albino mice. A distinctly increased level of lipid peroxide (LPO) free radicals along with the endogenous hepatic enzymes were evident in chromium induced hepatotoxicity in mice. However, oligosaccharides treatment mitigated these effects by reducing the LPO production and increasing ALT, ALP, and AST levels, probably due to relieving the oxidative stress. DNA fragmentation assays illustrated that Cr(VI) exposure induced massive apoptosis in liver by damaging the DNA which was then remediated by oligosaccharides supplementation. Histopathological observations confirmed that the oligosaccharide treatment reverses the architectural changes in liver induced by chromium. These results suggest that oligosaccharides obtained from cress seed mucilage polysaccharides through P. pastoris fermentation ameliorate the oxidative stress and apoptosis and act as hepatoprotective agent against chromium-induced liver injury.

Promoters in Pichia pastoris: A Toolbox for Fine-Tuned Gene Expression.

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential.

Expression, characterization, and application of human-like recombinant gelatin.

Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. With irreplaceable biological functions in the field of biomedical science and tissue engineering, it has been widely applied. The amino acid sequence of recombinant human-like gelatin was constructed through a newly designed hexamer composed of six protein monomer sequences in series, with the minimum repeating unit being the characteristic Gly-X-Y sequence found in type III human collagen α1 chain. The nucleotide sequence was subsequently inserted into the genome of Pichia pastoris to enable soluble secretion expression of recombinant gelatin. At the shake flask fermentation level, the yield of recombinant gelatin is up to 0.057 g/L, and its purity can rise up to 95% through affinity purification. It was confirmed in the molecular weight determination and amino acid analysis that the amino acid composition of the obtained recombinant gelatin is identical to that of the theoretically designed. Furthermore, scanning electron microscopy revealed that the freeze-dried recombinant gelatin hydrogel exhibited a porous structure. After culturing cells continuously within these gelatin microspheres for two days followed by fluorescence staining and observation through confocal laser scanning microscopy, it was observed that cells clustered together within the gelatin matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant gelatin as a biomedical material.

Metabolic heat based specific growth rate estimators: Does the choice of estimation model influence the state of bioprocesses?

Accurate and reliable estimation of specific growth rate (µ) in real-time is pivotal for reliable process monitoring of a bioprocess and subsequent implementation of advanced control strategies. Gibbs free energy dissipation is imminent for any biological system, and the metabolic heat flow measurements (calorimetry) formed the basis for estimating µ. However, the rationale behind selecting a suitable µ estimator model based on calorimetric perspective remains unexplored. The present investigation addresses the notion behind the selection of an appropriate estimator for µ and the assessment of the estimator models was illustrated using different types of energy metabolism, namely, high exothermic and low exothermic processes. The results indicated that the µ values from the instantaneous heat flow estimator significantly deviated (10-fold higher) from the offline values for highly exothermic process. Notably, the cumulative heat-based estimator accurately estimated µ values on both types of energy metabolism with performance metrics <0.005 h-1.

dMAD7 is a promising tool for targeted gene regulation in the methylotrophic yeast Komagataella phaffii.

The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression.

Pathway reconstruction and metabolic engineering for the de novo and enhancing production of monacolin J in Pichia pastoris.

The statin is the primary cholesterol-lowering drug. Monacolin J (MJ) is a key intermediate in the biosynthetic pathway of statin. It was obtained in industry by the alkaline hydrolysis of lovastatin. The hydrolysis process resulted in multiple by-products and expensive cost of wastewater treatment. In this work, we used Pichia pastoris as the host to produce the MJ. The biosynthesis pathway of MJ was built in P. pastoris. The stable recombinant strain MJ2 was obtained by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 genome-editing tool, and produced the MJ titer of 153.6 ± 2.4 mg/L. The metabolic engineering was utilized to enhance the production of MJ, and the fermentation condition was optimized. The MJ titer of 357.5 ± 5.0 mg/L was obtained from the recombinant strain MJ5-AZ with ATP-dependent citrate lyase (ACL), glucose-6-phosphate dehydrogenase (ZWF1) and four lovB genes, 132.7% higher than that from the original strain MJ2. The recombinant strain MJ5-AZ was cultured in a 7-L fermenter, and the MJ titer of 1493.0 ± 9.2 mg/L was achieved. The results suggested that increasing the gene dosage of rate-limiting step in the biosynthesis pathway of chemicals could improve the titer of production. It might be applicable to the production optimization of other polyketide metabolites.