Dynamically Regulating Homologous Recombination Enables Precise Genome Editing in Ogataea polymorpha.

Methylotrophic yeast Ogataea polymorpha has become a promising cell factory due to its efficient utilization of methanol to produce high value-added chemicals. However, the low homologous recombination (HR) efficiency in O. polymorpha greatly hinders extensive metabolic engineering for industrial applications. Overexpression of HR-related genes successfully improved HR efficiency, which however brought cellular stress and reduced chemical production due to constitutive expression of the HR-related gene. Here, we engineered an HR repair pathway using the dynamically regulated gene ScRAD51 under the control of the l-rhamnose-induced promoter PLRA3 based on the previously constructed CRISPR-Cas9 system in O. polymorpha. Under the optimal inducible conditions, the appropriate expression level of ScRAD51 achieved up to 60% of HR rates without any detectable influence on cell growth in methanol, which was 10-fold higher than that of the wild-type strain. While adopting as the chassis strain for bioproductions, the dynamically regulated recombination system had 50% higher titers of fatty alcohols than that static regulation system. Therefore, this study provided a feasible platform in O. polymorpha for convenient genetic manipulation without perturbing cellular fitness.

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.

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.

Engineering yeast for high-level production of ß-farnesene from sole methanol.

Methanol, a rich one-carbon feedstock, can be massively produced from CO2 by the liquid sunshine route, which is helpful to realize carbon neutrality. ß-Farnesene is widely used in the production of polymers, surfactants, lubricants, and also serves as a suitable substitute for jet fuel. Constructing an efficient cell factory is a feasible approach for ß-farnesene production through methanol biotransformation. Here, we extensively engineered the methylotrophic yeast Ogataea polymorpha for the efficient bio-production of ß-farnesene using methanol as the sole carbon source. Our study demonstrated that sufficient supply of precursor acetyl-CoA and cofactor NADPH in an excellent yeast chassis had a 1.3-fold higher ß-farnesene production than that of wild-type background strain. Further optimization of the mevalonate pathway and enhancement of acetyl-CoA supply led to a 7-fold increase in ß-farnesene accumulation, achieving the highest reported sesquiterpenoids production (14.7 g/L with a yield of 46 mg/g methanol) from one-carbon feedstock under fed-batch fermentation in bioreactor. This study demonstrates the great potential of engineering O. polymorpha for high-level terpenoid production from methanol.

Engineering Yeast Peroxisomes for α-Bisabolene Production from Sole Methanol with the Aid of Proteomic Analysis.

Microbial metabolic engineering provides a feasible approach to sustainably produce advanced biofuels and biochemicals from renewable feedstocks. Methanol is an ideal feedstock since it can be massively produced from CO2 through green energy, such as solar energy. However, engineering microbes to transform methanol and overproduce chemicals is challenging. Notably, the microbial production of isoprenoids from methanol is still rarely reported. Here, we extensively engineered Pichia pastoris (syn. Komagataella phaffii) for the overproduction of sesquiterpene α-bisabolene from sole methanol by optimizing the mevalonate pathway and peroxisomal compartmentalization. Furthermore, through label-free quantification (LFQ) proteomic analysis of the engineered strains, we identified the key bottlenecks in the peroxisomal targeting pathway, and overexpressing the limiting enzyme EfmvaE significantly improved α-bisabolene production to 212 mg/L with the peroxisomal pathway. The engineered strain LH122 with the optimized peroxisomal pathway produced 1.1 g/L α-bisabolene under fed-batch fermentation in shake flasks, achieving a 69% increase over that of the cytosolic pathway. This study provides a viable approach for overproducing isoprenoid from sole methanol in engineered yeast cell factories and shows that proteomic analysis can help optimize the organelle compartmentalized pathways to enhance chemical production.

Ogataea polymorpha as a next-generation chassis for industrial biotechnology.

Ogataea (Hansenula) polymorpha is a nonconventional yeast with some unique characteristics, including fast growth, thermostability, and broad substrate spectrum. Other than common applications for protein production, O. polymorpha is attracting interest for chemical and protein production from methanol; a promising feedstock for the next-generation biomanufacturing due to its abundant sources and excellent characteristics. Benefiting from the development of synthetic biology, it has been engineered to produce value-added chemicals by extensively rewiring cellular metabolism. This Review discusses recently developed synthetic biology tools of O. polymorpha. The advances of chemicals production and systems biology were reviewed comprehensively. Finally, we look ahead to the developments of biomanufacturing in O. polymorpha to make an overall understanding of this chassis for academia and industry.

Synthetic biology for Taxol biosynthesis and sustainable production.

Incomplete understanding of the biosynthetic pathway of the anticancer compound Taxol hinders its production by metabolic engineering. Recent reports by Jiang et al. and other groups now describe the missing steps in Taxol biosynthesis, notably including oxetane ring formation. These findings will promote the sustainable production of Taxol through synthetic biology.

Promoter engineering enables precise metabolic regulation towards efficient ß-elemene production in Ogataea polymorpha.

Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production. However, promoters in nonconventional yeasts such as Ogataea polymorpha are always limited, which results in incompatible gene modulation. Here, we expanded the promoter library in O. polymorpha based on transcriptional data, among which 13 constitutive promoters had the strengths ranging from 0-55% of PGAP, the commonly used strong constitutive promoter, and 2 were growth phase-dependent promoters. Subsequently, 2 hybrid growth phase-dependent promoters were constructed and characterized, which had 2-fold higher activities. Finally, promoter engineering was applied to precisely regulate cellular metabolism for efficient production of ß-elemene. The glyceraldehyde-3-phosphate dehydrogenase gene GAP was downregulated to drive more flux into pentose phosphate pathway (PPP) and then to enhance the supply of acetyl-CoA by using phosphoketolase-phosphotransacetylase (PK-PTA) pathway. Coupled with the phase-dependent expression of synthase module (ERG20∼LsLTC2 fusion), the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose) was achieved in strain YY150U under fed-batch fermentation in shake flasks. This work characterized and engineered a series of promoters, that can be used to fine-tune genes for constructing efficient yeast cell factories.

De novo biosynthesis of the hops bioactive flavonoid xanthohumol in yeast.

The flavonoid xanthohumol is an important flavor substance in the brewing industry that has a wide variety of bioactivities. However, its unstable structure results in its low content in beer. Microbial biosynthesis is considered a sustainable and economically viable alternative. Here, we harness the yeast Saccharomyces cerevisiae for the de novo biosynthesis of xanthohumol from glucose by balancing the three parallel biosynthetic pathways, prenyltransferase engineering, enhancing precursor supply, constructing enzyme fusion, and peroxisomal engineering. These strategies improve the production of the key xanthohumol precursor demethylxanthohumol (DMX) by 83-fold and achieve the de novo biosynthesis of xanthohumol in yeast. We also reveal that prenylation is the key limiting step in DMX biosynthesis and develop tailored metabolic regulation strategies to enhance the DMAPP availability and prenylation efficiency. Our work provides feasible approaches for systematically engineering yeast cell factories for the de novo biosynthesis of complex natural products.

An improved CRISPRi system in Pichia pastoris.

CRISPR interference (CRISPRi) has been developed and widely used for gene repression in various hosts. Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor domains. The CRISPRi system shows strong repression of eGFP, with the highest efficiency of 85%. Repression of native genes is demonstrated by targeting AOX1 promoter. AOX1 is efficiently repressed and the mutant strains show much slower growth in methanol medium. Effects of gRNA expression and processing on CRISPRi efficiency is also investigated. It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression, and Csy4 cleavage shows higher repression efficiency. However, gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of eGFP. By expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites, both genes can be repressed simultaneously. Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes (FAA1 and FAA2). Both genes are efficiently repressed, demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in P. pastoris.

Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose.

Bio-refining lignocellulose could provide a sustainable supply of fuels and fine chemicals; however, the challenges associated with the co-utilization of xylose and glucose typically compromise the efficiency of lignocellulose conversion. Here we engineered the industrial yeast Ogataea polymorpha (Hansenula polymorpha) for lignocellulose biorefinery by facilitating the co-utilization of glucose and xylose to optimize the production of free fatty acids (FFAs) and 3-hydroxypropionic acid (3-HP) from lignocellulose. We rewired the central metabolism for the enhanced supply of acetyl-coenzyme A and nicotinamide adenine dinucleotide phosphate hydrogen, obtaining 30.0 g l-1 of FFAs from glucose, with productivity of up to 0.27 g l-1 h-1. Strengthening xylose uptake and catabolism promoted the synchronous utilization of glucose and xylose, which enabled the production of 38.2 g l-1 and 7.0 g l-1 FFAs from the glucose-xylose mixture and lignocellulosic hydrolysates, respectively. Finally, this efficient cell factory was metabolically transformed for 3-HP production with the highest titer of 79.6 g l-1 in fed-batch fermentation in mixed glucose and xylose.

Construction and Optimization of Nonclassical Isoprenoid Biosynthetic Pathways in Yeast Peroxisomes for (+)-Valencene Production.

Isoprenoids are a kind of natural product with various activities, but their plant extraction suffers low concentration. The rapid development of synthetic biology offers a sustainable route for supply of high-value-added natural products by engineering microorganisms. However, the complexity of cellular metabolism makes engineering endogenous isoprenoid biosynthetic pathways with metabolic interaction difficult. Here, for the first time, we constructed and optimized three types of isoprenoid pathways (the Haloarchaea-type, Thermoplasma-type, and isoprenoid alcohol pathway) in yeast peroxisomes for the synthesis of sesquiterpene (+)-valencene. In yeast, the Haloarchaea-type MVA pathway is more effective than the classical MVA pathway. MVK and IPK were determined to be the rate-limiting steps of the Haloarchaea-type MVA pathway, and the production of 869 mg/L (+)-valencene under fed-batch fermentation in shake flasks was realized. This work expands isoprenoid synthesis in eukaryotes and provides a more efficient pathway for isoprenoid synthesis.

Peroxisomal metabolic coupling improves fatty alcohol production from sole methanol in yeast.

Methanol is an ideal feedstock for chemical and biological manufacturing. Constructing an efficient cell factory is essential for producing complex compounds through methanol biotransformation, in which coordinating methanol use and product synthesis is often necessary. In methylotrophic yeast, methanol utilization mainly occurs in peroxisomes, which creates challenges in driving the metabolic flux toward product biosynthesis. Here, we observed that constructing the cytosolic biosynthesis pathway resulted in compromised fatty alcohol production in the methylotrophic yeast Ogataea polymorpha. Alternatively, peroxisomal coupling of fatty alcohol biosynthesis and methanol utilization significantly improved fatty alcohol production by 3.9-fold. Enhancing the supply of precursor fatty acyl-CoA and cofactor NADPH in the peroxisomes by global metabolic rewiring further improved fatty alcohol production by 2.5-fold and produced 3.6 g/L fatty alcohols from methanol under fed-batch fermentation. We demonstrated that peroxisome compartmentalization is helpful for coupling methanol utilization and product synthesis, and with this approach, constructing efficient microbial cell factories for methanol biotransformation is feasible.

Global metabolic rewiring of the nonconventional yeast Ogataea polymorpha for biosynthesis of the sesquiterpenoid ß-elemene.

Bioproduction of natural products via microbial cell factories is a promising alternative to traditional plant extraction. Recently, nonconventional microorganisms have emerged as attractive chassis hosts for biomanufacturing. One such microorganism, Ogataea polymorpha is an industrial yeast used for protein expression with numerous advantages, such as thermal-tolerance, a wide substrate spectrum and high-density fermentation. Here, we systematically rewired the cellular metabolism of O. polymorpha to achieve high-level production of the sesquiterpenoid ß-elemene by optimizing the mevalonate pathway, enhancing the supply of NADPH and acetyl-CoA, and downregulating competitive pathways. The engineered strain produced 509 mg/L and 4.7 g/L of ß-elemene under batch and fed-batch fermentation, respectively. Therefore, this study identified the potential industrial application of O. polymorpha as a good microbial platform for producing sesquiterpenoids.

Auxotrophs compromise cell growth and fatty acid production in Saccharomyces cerevisiae.

Auxotrophic marker genes have been widely used for genetic engineering in yeast. However, the effects of amino acids or nucleotides deficiency in auxotrophic strains on cell growth and product synthesis were rarely reported. In this study, a total of eight auxotrophic strains of Saccharomyces cerevisiae with single knockout of selection markers were obtained. Cell growth and free fatty acid (FFA) production of these auxotrophic strains were evaluated with supplementation of different concentrations of amino acids or nucleotides. Generally, except ade2Δ mutants, most auxotrophic strains showed decreased cell growth and FFA production, which could be recovered by adding higher concentrations of supplements. LEU2 deletion (leu2Δ) damaged both cell growth and FFA production even with supplementation of 1000 mg L-1 leucine. This study shows that growth and product biosynthesis of auxotrophs could be limited by insufficient supplementation of amino acids or nucleotides, and provides guidance on supplementation of these nutrients during fermentation to maximize cell growth and product biosynthesis.

Skeleton-Reorganizing Coupling Reactions of Cycloheptatriene and Cycloalkenones with Amines.

Skeletal reorganization reactions have emerged as an intriguing tool for converting readily available compounds into complicated molecules inaccessible by traditional methods. Herein, we report a unique skeleton-reorganizing coupling reaction of cycloheptatriene and cycloalkenones with amines. In the presence of Rh/acid catalysis, cycloheptatriene can selectively couple with anilines to deliver fused 1,2-dihydroquinoline products. Mechanistic studies indicate that the retro-Mannich type ring-opening and subsequent intramolecular Povarov reaction account for the ring reorganization. Our mechanistic studies also revealed that skeleton-reorganizing amination between anilines and cycloalkenones can be achieved with acid. The synthetic utilization of this skeleton-reorganizing coupling reaction was showcased with a gram-scale reaction, synthetic derivatizations, and the late-stage modification of commercial drugs.

Engineering yeast for high-level production of diterpenoid sclareol.

The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production.

Spatial-temporal regulation of fatty alcohol biosynthesis in yeast.

BACKGROUND: Construction of efficient microbial cell factories is one of the core steps for establishing green bio-manufacturing processes. However, the complex metabolic regulation makes it challenging in driving the metabolic flux toward the product biosynthesis. Dynamically coupling the biosynthetic pathways with the cellular metabolism at spatial-temporal manner should be helpful for improving the production with alleviating the cellular stresses. RESULTS: In this study, we observed the mismatch between fatty alcohol biosynthesis and cellular metabolism, which compromised the fatty alcohol production in Saccharomyces cerevisiae. To enhance the fatty alcohol production, we spatial-temporally regulated fatty alcohol biosynthetic pathway by peroxisomal compartmentalization (spatial) and dynamic regulation of gene expression (temporal). In particular, fatty acid/acyl-CoA responsive promoters were identified by comparative transcriptional analysis, which helped to dynamically regulate the expression of acyl-CoA reductase gene MaFAR1 and improved fatty alcohol biosynthesis by 1.62-fold. Furthermore, enhancing the peroxisomal supply of acyl-CoA and NADPH further improved fatty alcohol production to 282 mg/L, 2.52 times higher than the starting strain. CONCLUSIONS: This spatial-temporal regulation strategy partially coordinated fatty alcohol biosynthesis with cellular metabolism including peroxisome biogenesis and precursor supply, which should be applied for production of other products in microbes.

Construction of microbial chassis for terpenoid discovery.

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Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris.

BACKGROUND: The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts. RESULTS: The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number. CONCLUSIONS: Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.