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.

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.

Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast.

Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-L-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l-1) and ferulic acid (3.8 ± 0.3 g l-1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.

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.

Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts.

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha in metabolic engineeringare expounded. • Three promoter engineering strategies are introduced in order to expand the promoter toolbox.

Characterizing methanol metabolism-related promoters for metabolic engineering of Ogataea polymorpha.

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.

CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications.

Non-conventional yeasts are playing important roles as cell factories for bioproduction of biofuels, food additives and proteins with outstanding natural characteristics. However, the precise genome editing is challenging in non-conventional yeasts due to lack of efficient genetic tools. In the past few years, CRISPR-based genome editing worked as a revolutionary tool for genetic engineering and showed great advantages in cellular metabolic engineering. Here, we review the current advances and barriers of CRISPR-Cas9 for genome editing in non-conventional yeasts and propose the possible solutions in enhancing its efficiency for precise genetic engineering.

Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

General Control Nonderepressible 2 (GCN2) Kinase Inhibits Target of Rapamycin Complex 1 in Response to Amino Acid Starvation in Saccharomyces cerevisiae.

In eukaryotic cells, two conserved protein kinases, Gcn2 and TOR complex 1 (TORC1), couple amino acid conditions to protein translation. Gcn2 functions as an amino acid sensor and is activated by uncharged tRNAs that accumulate when intracellular amino acids are limited. Activated Gcn2 phosphorylates and inhibits eukaryotic initiation factor-2α (eIF2α), resulting in repression of general protein synthesis. Like Gcn2, TORC1 is also involved in sensing amino acid conditions. However, the underlying mechanism remains unclear. In the present study, we show that TORC1 is a direct target of Gcn2 kinase in the yeast Saccharomyces cerevisiae In response to amino acid starvation, Gcn2 binds to TORC1 and phosphorylates Kog1, the unique regulatory subunit of TORC1, resulting in down-regulation of TORC1 kinase activity. In the absence of Gcn2, TORC1 signaling activity increases and becomes unresponsive to amino acid starvation. Our findings demonstrate that TORC1 is an effector of Gcn2 in amino acid signaling, hence defining a novel mechanism by which TORC1 senses amino acid starvation.

Characterization of inulinase promoter from Kluyveromyces marxianus for intensive protein expression in industrial biotechnology.

Inulinase from Kluyveromyces marxianus (KmINU1) has wide application in industrial biotechnology, and it is supposed that its expression is regulated at the transcriptional level via the promoter. Therefore, in this study, the KmINU1 promoter, fused to the reporter EGFP gene, was analyzed to determine its fundamental regulatory mechanisms in Saccharomyces cerevisiae S288C (ATCC 204508). Induction by inulin and repression by high glucose concentrations of KmINU1 promoters are reported, and the promoter with a length of 353 bp was shown to have the highest strength with the weakest responses to high glucose concentration. Responses of the KmINU1 promoter to carbon source were shown to be related to the Mig1 binding site extending from -496 to -485 bp. Hence, we propose clear regulation mechanisms of the inulinase promoter, which may provide a powerful theoretical reference for its application in protein production.

Synergistic effect of thioredoxin and its reductase from Kluyveromyces marxianus on enhanced tolerance to multiple lignocellulose-derived inhibitors.

BACKGROUND: Multiple lignocellulose-derived inhibitors represent great challenges for bioethanol production from lignocellulosic materials. These inhibitors that are related to the levels of intracellular reactive oxidative species (ROS) make oxidoreductases a potential target for an enhanced tolerance in yeasts. RESULTS: In this study, the thioredoxin and its reductase from Kluyveromyces marxianus Y179 was identified, which was subsequently achieved over-expression in Saccharomyces cerevisiae 280. In spite of the negative effects by expression of thioredoxin gene (KmTRX), the thioredoxin reductase (KmTrxR) helped to enhance tolerance to multiple lignocellulose-derived inhibitors, such as formic acid and acetic acid. In particular, compared with each gene expression, the double over-expression of KmTRX2 and KmTrxR achieved a better ethanol fermentative profiles under a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. At last, the mechanism that improves the tolerance depended on a normal level of intracellular ROS for cell survival under stress. CONCLUSIONS: The synergistic effect of KmTrxR and KmTRX2 provided the potential possibility for ethanol production from lignocellulosic materials, and give a general insight into the possible toxicity mechanisms for further theoretical research.

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.

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.

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.

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.

[Production of fatty acids by engineered Ogataea polymorpha].

Fatty acids (FA) are widely used as feed stocks for the production of cosmetics, personal hygiene products, lubricants and biofuels. Ogataea polymorpha is considered as an ideal chassis for bio-manufacturing, due to its outstanding characteristics such as methylotroph, thermal-tolerance and wide substrate spectrum. In this study, we harnessed O. polymorpha for overproduction of fatty acids by engineering its fatty acid metabolism and optimizing the fermentation process. The engineered strain produced 1.86 g/L FAs under the optimized shake-flask conditions (37℃, pH 6.4, a C/N ratio of 120 and an OD600 of seed culture of 6-8). The fed-batch fermentation process was further optimized by using a dissolved oxygen (DO) control strategy. The C/N ratio of initial medium was 17.5, and the glucose medium with a C/N ratio of 120 was fed when the DO was higher than 30%. This operation resulted in a titer of 18.0 g/L FA, indicating the potential of using O. polymorpha as an efficient cell factory for the production of FA.

Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol.

Methanol is an ideal feedstock for biomanufacturing that can be beneficial for global carbon neutrality; however, the toxicity of methanol limits the efficiency of methanol metabolism toward biochemical production. We here show that engineering production of free fatty acids from sole methanol results in cell death with decreased cellular levels of phospholipids in the methylotrophic yeast Ogataea polymorpha, and cell growth is restored by adaptive laboratory evolution. Whole-genome sequencing of the adapted strains reveals that inactivation of LPL1 (encoding a putative lipase) and IZH3 (encoding a membrane protein related to zinc metabolism) preserve cell survival by restoring phospholipid metabolism. Engineering the pentose phosphate pathway and gluconeogenesis enables high-level production of free fatty acid (15.9 g l-1) from sole methanol. Preventing methanol-associated toxicity underscores the link between lipid metabolism and methanol tolerance, which should contribute to enhancing methanol-based biomanufacturing.

Overproduction of 3-hydroxypropionate in a super yeast chassis.

3-Hydroxypropionate (3-HP) is a platform chemical for production of acrylic acid, acrylamide and biodegradable polymers. Several microbial cell factories have been constructed for production of 3-HP from malonyl-CoA by using a malonyl-CoA reductase, which however suffer from inadequate supply of precursor and cofactor. Here 3-HP biosynthesis was optimized in a super yeast chassis with sufficient supply of precursor malonyl-CoA and cofactor NADPH, which had a 3-fold higher 3-HP (1.4 g/L) than that of wild-type background. The instability of the engineered strain was observed in fed-batch fermentation due to the plasmid loss, which may be caused by the toxic intermediate malonate semialdehyde. Genome integration of MCR-C encoding C-terminal of MCR enabled stable gene expression and much higher 3-HP production of 4.4 g/L under batch fermentation and 56.5 g/L under fed-batch fermentation with a yield of 0.31 g/g glucose. This was the highest 3-HP production reported from glucose in engineered microbes.

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.