Transcription Factor Pdr3p Promotes Carotenoid Biosynthesis by Activating GAL Promoters in Saccharomyces cerevisiae.

Pleiotropic drug resistance (PDR) family proteins have been extensively studied for their roles in transporting hydrophobic substances, including carotenoids. Overexpression of the PDR family regulator Pdr3p was recently found to boost the biosynthesis of carotenoids, which could not be explained by enhanced product secretion due to the meager extracellular proportions. To provide insights into the possible mechanism, comparative transcriptomics, reverse metabolic engineering, and electrophoretic mobility shift assay (EMSA) were conducted. Transcriptomic data suggested an unexpected correlation between Pdr3p overexpression and the transcriptional levels of GAL promoter-driven genes. This assumption was verified using mCherry and the lycopene synthetic pathway as the reporters. qRT-PCR and EMSA provided further evidence for the activation of GAL promoters by Pdr3p binding to their upstream activation sequences (UASs). This work gives insight into the mechanism of Pdr3p-promoted carotenoid production and highlights the complicated metabolic networking between transcriptional factors and promoters in yeast.

XylR Overexpression in Escherichia coli Alleviated Transcriptional Repression by Arabinose and Enhanced Xylitol Bioproduction from Xylose Mother Liquor.

Xylitol is a polyol widely used in food, pharmaceuticals, and light industries. It is currently produced through the chemical catalytic hydrogenation of xylose and generates xylose mother liquor as a substantial byproduct in the procedure of xylose extraction. If xylose mother liquor could also be efficiently bioconverted to xylitol, the greenness and atom economy of xylitol production would be largely improved. However, xylose mother liquor contains a mixture of glucose, xylose, and arabinose, raising the issue of carbon catabolic repression in its utilization by microbial conversion. Targeting this challenge, the transcriptional activator XylR was overexpressed in a previously constructed xylitol-producing E. coli strain CPH. The resulting strain CPHR produced 16.61 g/L of xylitol in shake-flask cultures from the mixture of corn cob hydrolysate and xylose mother liquor (1:1, v/v) with a xylose conversion rate of 90.1%, which were 2.23 and 2.15 times higher than the starting strain, respectively. Furthermore, XylR overexpression upregulated the expression levels of xylE, xylF, xylG, and xylH genes by 2.08-2.72 times in arabinose-containing medium, suggesting the alleviation of transcriptional repression of xylose transport genes by arabinose. This work lays the foundation for xylitol bioproduction from xylose mother liquor.

Rational design of transaminases based on comparative analysis of catalytically active and distance-free modes of the high-energy intermediate state.

Bioproduction of chiral amines is limited by low transaminase (TA) activity on nonnatural substrates, leading to the need for protein engineering. To address the challenge of quickly and precisely identifying the engineering targets, a strategy was proposed based on analyzing the mode changes in the high-energy intermediate state (H-state) of the substrate-enzyme complex during catalysis. By substituting the residues with minimal structural changes in catalytically active mode (A-mode) and distance-free mode (F-mode) of the H-state complex with more conserved ones to stabilize it, a TA mutant M5(T295C/L387A/V436A) with 121.9-fold higher activity for synthesizing the (S)-Rivastigmine precursor (S)-1-(3-methoxyphenyl)ethylamine was created. The applicability of this strategy was also validated by engineering another TA for 1.52-fold higher activity and >99% selectivity toward (R)-3-amino-1-butanol biopreparation. The much higher stereoselectivity of the mutant compared with the wild type (28.3%) demonstrated that this strategy is not only advantageous in engineering enzyme activity but also applicable for modulating stereoselectivity.

Efficient Secretory Production of δ-Tocotrienol by Combining Pathway Modularization and Transportation Engineering.

The vitamin E component δ-tocotrienol has shown impressive activities in radioprotection, neuroprotection, and cholesterol reduction. Its production is limited by the low content in plants and difficulty in separation from other tocotrienols. Fermentative production using a microbial cell factory that exclusively produces and secretes δ-tocotrienol is a promising alternative approach. Assembly of the δ-tocotrienol synthetic pathway in Saccharomyces cerevisiae followed by comprehensive pathway engineering led to the production of 73.45 mg/L δ-tocotrienol. Subsequent addition of 2-hydroxypropyl-ß-cyclodextrin (CD) and overexpression of the transcription factor PDR1 significantly elevated δ-tocotrienol titer to 241.7 mg/L (63.65 mg/g dry cell weight) in shake flasks, with 30.4% secreted. By properly adding CD and the in situ extractant olive oil, 181.12 mg/L of δ-tocotrienol was collected as an extracellular product, accounting for 85.6% of the total δ-tocotrienol production. This process provides not only a promising δ-tocotrienol cell factory but also insights into yeast engineering toward secretory production of other terpenoids.

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies.

Terpenoids are a diverse group of compounds with isoprene units as basic building blocks. They are widely used in the food, feed, pharmaceutical, and cosmetic industries due to their diverse biological functions such as antioxidant, anticancer, and immune enhancement. With an increase in understanding the biosynthetic pathways of terpenoids and advances in synthetic biology techniques, microbial cell factories have been built for the heterologous production of terpenoids, with the oleaginous yeast Yarrowia lipolytica emerging as an outstanding chassis. In this paper, recent progress in the development of Y. lipolytica cell factories for terpenoid production with a focus on the advances in novel synbio tools and metabolic engineering strategies toward enhanced terpenoid biosynthesis is reviewed.

Improved Bioproduction of the Nylon 12 Monomer by Combining the Directed Evolution of P450 and Enhancing Heme Synthesis.

The nylon 12 (PA12) monomer ω-aminododecanoic acid (ω-AmDDA) could be synthesized from lauric acid (DDA) through multi-enzyme cascade transformation using engineered E. coli, with the P450 catalyzing terminal hydroxylation of DDA as a rate-limiting enzyme. Its activity is jointly determined by the heme domain and the reductase domain. To obtain a P450 mutant with higher activity, directed evolution was conducted using a colorimetric high-throughput screening (HTS) system with DDA as the real substrate. After two rounds of directed evolution, a positive double-site mutant (R14R/D629G) with 90.3% higher activity was obtained. Molecular docking analysis, kinetic parameter determination and protein electrophoresis suggested the improved soluble expression of P450 resulting from the synonymous mutation near the N-terminus and the shortened distance of the electron transfer between FMN and FAD caused by D629G mutation as the major reasons for activity improvement. The significantly increased kcat and unchanged Km provided further evidence for the increase in electron transfer efficiency. Considering the important role of heme in P450, its supply was strengthened by the metabolic engineering of the heme synthesis pathway. By combining P450-directed evolution and enhancing heme synthesis, 2.02 ± 0.03 g/L of ω-AmDDA was produced from 10 mM DDA, with a yield of 93.6%.

Hierarchical dynamic regulation of Saccharomyces cerevisiae for enhanced lutein biosynthesis.

Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.

Spatiotemporal Regulation of Astaxanthin Synthesis in S. cerevisiae.

As a high-valued antioxidant, astaxanthin biosynthesis using microbial cell factories has attracted increasing attention. However, its lipophilic nature conflicts with the limited storage capacity for lipophilic substances of model microorganisms such as Saccharomyces cerevisiae. Expansion of lipid droplets by enhancing lipid synthesis provides more storage room while diverting the metabolic flux from the target pathway. Therefore, proper spatial regulation is required. In this study, a library of genes related to lipid metabolism were screened using the trifunctional CRISPR system, identifying opi3 and hrd1 as new engineering targets to promote astaxanthin synthesis by moderately rather than excessively upregulating lipid synthesis. The astaxanthin yield reached 9.79 mg/g DCW after lipid engineering and was further improved to 10.21 mg/g DCW by balancing the expression of ß-carotene hydroxylase and ketolase. Finally, by combining spatial regulation through lipid droplet engineering and temporal regulation via temperature-responsive pathway expression, 446.4 mg/L astaxanthin was produced in fed-batch fermentation.

Production of retinoic acid by engineered Saccharomyces cerevisiae using an endogenous aldehyde dehydrogenase.

Retinoic acid (RA), a vitamin A (retinol)-derived lipophilic compound, is involved in various physiological functions. The demand for RA is growing in the pharmaceutical industry, but RA biosynthesis is still in its infancy compared to other forms of retinoids such as retinol and retinal, largely due to the lack of efficient retinal dehydrogenases. To achieve effective biosynthesis of RA, the catalytic activities of exogenous retinal dehydrogenases were comparatively analyzed in a previously constructed retinoids-producing Saccharomyces cerevisiae strain, followed by mining of endogenous enzymes with higher retinal dehydrogenase activities using homology-based search. After confirming the retinal oxidation activity of the endogenous aldehyde dehydrogenase Hfd1 using in vivo and in vitro experiments, it was overexpressed in multiple copies, and the resulting strain produced 99.71 mg/L of RA in shake-flask cultures. Finally, 545.28 mg/L of RA was produced in fed-batch fermentation. This study suggests the yeast endogenous Hfd1 as a potent catalyst for RA biosynthesis, and demonstrates the potential of yeast as a platform for RA production.

Alleviation of the Byproducts Formation Enables Highly Efficient Biosynthesis of Rosmarinic Acid in Saccharomyces cerevisiae.

Rosmarinic acid as a polyphenolic compound has great values in the pharmaceutical, cosmetic, and food industries. To achieve efficient biosynthesis of rosmarinic acid, the major obstacles such as imbalanced metabolic flux among branching pathways and substrate promiscuity of pathway enzymes should be eliminated. Here, a rosmarinic acid producing Saccharomyces cerevisiae strain was constructed by introducing codon optimized d-lactate dehydrogenase gene mutant (OD-LDHY52A), 4-coumarate CoA ligase gene (OPc4CL2), and rosmarinic acid synthase gene (OMoRAS) into a previously constructed caffeic acid hyper-producer. To identify the metabolic bottleneck, the substrate specificity of OPc4CL2 and OMoRAS was figured out by bioconversion experiments and HPLC-MS/MS analysis. Subsequently, the byproducts formation was alleviated by removing prephenate dehydratase and tuning down the expression level of OPc4CL2. The final strain YRA113-15B produced 208 mg/L rosmarinic acid in a shake-flask culture (a 63-fold improvement over the initial strain), which was the highest rosmarinic acid titer by engineered microbial cells reported to date. This work provides a promising platform for fermentative production of rosmarinic acid and offers a strategy to overcome the intrapathway competition.

Recent advances in construction and regulation of yeast cell factories.

The past decade has witnessed the rapid progress in development of synthetic biology, and advances in construction of yeast cell factories open vast opportunities for green and sustainable production of chemicals. Focusing on the progress in yeast engineering for production of plant natural products in the last 5 years, this review introduces different yeast chassis used for cell factory construction, including Saccharomyces cerevisiae, Yarrowia lipolytica and Komagataella phaffii, together with the emerging genome editing tools. The metabolic regulation strategies developed for yeast engineering are highlighted, such as subcellular pathway localization dynamic regulation, and transporter engineering. C1-based chemical bioproduction by engineered yeast is also covered. Finally, the existing challenges and future prospects in creating efficient yeast cell factories are summarized.

Enhanced CoQ10 production by genome modification of Rhodobacter sphaeroides via Tn7 transposition.

As a native CoQ10 producer, Rhodobacter sphaeroides has been extensively engineered to enhance CoQ10 production. However, the genetic manipulations using plasmids suffer from risk of plasmid loss during propagation process, biomass impairment due to cellular burden and bio-safety concerns. In this paper, genomic manipulations via Tn7 transposition was conducted to boost the CoQ10 biosynthesis in R. sphaeroides. The titer production and content of CoQ10 were improved by 18.44% and 18.87%, respectively compared to the wild type, when an additional copy of dxs and dxr were integrated into the genome. Further overexpression of idi and ispD by genomic integration created strain RSPCDDII with CoQ10 production and content of 81.23 mg/L and 5.93 mg/g, which were 54.28 and 55.97% higher than those of the wild type. The gene segments were successfully inserted into the attTn7 site of the R. sphaeroides genome. Meanwhile, the biomass was not affected. Compared to overexpression of genes on plasmids, this strategy could enhance protein expression to a proper level without affecting cell growth, and in a more stable manner.

Secretory Production of Tocotrienols in Saccharomyces cerevisiae.

Tocotrienols as important components of vitamin E have attracted increasing attention, with recent progress made in their heterologous biosynthesis, but all as intracellular products. Aiming to further improve the tocotrienol production capacity of engineered yeast and to advance toward industrial fermentation of tocotrienols, we first optimized the synthetic pathway to enhance the tocotrienol yield and then attempted to realize their secretory production by exploring biphasic extractive fermentation conditions and screening for endogenous transporters. Finally, a Saccharomyces cerevisiae strain with tocotrienol yield of 25.57 mg/g dry cell weight was generated, and the tocotrienol titer reached 82.68 mg/L in shake-flask cultures, with 73.66% of the product secreted into the organic phase. For the first time, we have reported that the vitamin E components could be harvested as extracellular products of microbial cell factories, which could largely simplify the downstream process and could be of significance for fermentative production of these products.

Construction of Canthaxanthin-Producing Yeast by Combining Spatiotemporal Regulation and Pleiotropic Drug Resistance Engineering.

The ketocarotenoid canthaxanthin has important applications in the feed industry. Its biosynthesis using microbial cell factories is an attractive alternative to the current chemical synthesis route. Canthaxanthin-producing Saccharomyces cerevisiae was constructed by introducing the ß-carotene ketolase variant OBKTM29 into a ß-carotene producer. Subcellular re-localization of OBKTM29 was explored, together with copy number adjustment both in the cytoplasm and on the periplasmic membrane, to accelerate the conversion of ß-carotene to canthaxanthin. Moreover, pleiotropic drug resistance (PDR) regulators Pdr1 and Pdr3 were overexpressed to improve the stress tolerance of the yeast strain, leading to obviously enhanced canthaxanthin production. The synthetic pathway was then regulated by a temperature-responsive GAL system to separate product synthesis from cell growth. Finally, 1.44 g/L canthaxanthin was harvested in fed-batch fermentation. This work demonstrated the power of spatial and temporal regulation and the efficiency of PDR engineering in heterologous biosynthesis.

Enhanced thermal and alkaline stability of L-lysine decarboxylase CadA by combining directed evolution and computation-guided virtual screening.

As an important monomer for bio-based nylons PA5X, cadaverine is mainly produced by enzymatic decarboxylation of L-lysine. A key issue with this process is the instability of L-lysine decarboxylase (CadA) during the reaction due to the dissociation of CadA subunits with the accumulation of alkaline cadaverine. In this work, we attempted to improve the thermal and alkaline stability of CadA by combining directed evolution and computation-guided virtual screening. Interestingly, site 477 residue located at the protein surface and not the decamer interface was found as a hotspot in directed evolution. By combinatorial mutagenesis of the positive mutations obtained by directed evolution and virtual screening with the previously reported T88S mutation, K477R/E445Q/T88S/F102V was generated as the best mutant, delivering 37% improvement of cadaverine yield at 50 ºC and pH 8.0. Molecular dynamics simulations suggested the improved rigidity of regional structures, increased number of salt bridges, and enhancement of hydrogen bonds at the multimeric interface as possible origins of the improved stability of the mutant. Using this four-point mutant, 160.7 g/L of cadaverine was produced from 2.0 M Lysine hydrochloride at 50 °C without pH regulation, with a conversion of 78.5%, whereas the wild type produced 143.7 g/L cadaverine, corresponding to 70% conversion. This work shows the combination of directed evolution and virtual screening as an efficient protein engineering strategy.

Selective biosynthesis of retinol in S. cerevisiae.

The vitamin A component retinol has become an increasingly sought-after cosmetic ingredient. In previous efforts for microbial biosynthesis of vitamin A, a mixture of retinoids was produced. In order to efficiently produce retinol at high purity, the precursor and NADPH supply was first enhanced to improve retinoids accumulation in the S. cerevisiae strain constructed from a ß-carotene producer by introducing ß-carotene 15,15'-dioxygenase, following by screening of heterologous and endogenous oxidoreductases for retinal reduction. Env9 was found as an endogenous retinal reductase and its activity was verified in vitro. By co-expressing Env9 with the E. coli ybbO, as much as 443.43 mg/L of retinol was produced at 98.76% purity in bi-phasic shake-flask culture when the antioxidant butylated hydroxytoluene was added to prevent retinoids degradation. The retinol titer reached 2479.34 mg/L in fed-batch fermentation. The success in selective biosynthesis of retinol would lay a solid foundation for its biotechnological production.

Biotechnological advances for improving natural pigment production: a state-of-the-art review.

In current years, natural pigments are facing a fast-growing global market due to the increase of people's awareness of health and the discovery of novel pharmacological effects of various natural pigments, e.g., carotenoids, flavonoids, and curcuminoids. However, the traditional production approaches are source-dependent and generally subject to the low contents of target pigment compounds. In order to scale-up industrial production, many efforts have been devoted to increasing pigment production from natural producers, via development of both in vitro plant cell/tissue culture systems, as well as optimization of microbial cultivation approaches. Moreover, synthetic biology has opened the door for heterologous biosynthesis of pigments via design and re-construction of novel biological modules as well as biological systems in bio-platforms. In this review, the innovative methods and strategies for optimization and engineering of both native and heterologous producers of natural pigments are comprehensively summarized. Current progress in the production of several representative high-value natural pigments is also presented; and the remaining challenges and future perspectives are discussed.

Development of a Highly Efficient Copper-Inducible GAL Regulation System (CuIGR) in Saccharomyces cerevisiae.

Dynamic regulation of gene expression to decouple growth and production has been proven to be effective for improving the biosynthetic efficiency of microbial cell factories. However, the number of efficient regulatory systems available for regulation of Saccharomyces cerevisiae is limited. In the present study, a novel copper-inducible gene expression system (CuIGR) composed of the copper-induced transcriptional activator Gal4 and the copper-inhibited repressor Gal80 was constructed in S. cerevisiae. When Gal80 was fused with a N-degron tag (K15), the resulting CuIGR4 system exhibited the most stringent regulation of gene expression driven by GAL1/2/7/10 promoters. As compared to the native Cu2+-inducible CUP1 promoter, the CuIGR4 system amplified the response to copper by as much as 2.7 folds, resulting in 72-fold induction of EGFP expression and a 33-fold change in lycopene production (3-100 mg/L) with addition of 20 µM copper. This newly developed copper-inducible system provides a powerful tool for gene expression control in S. cerevisiae, which is expected to be widely applicable in the regulation of yeast cell factories for enhanced biosynthesis of valuable products.

[Advances in metabolic engineering of Saccharomyces cerevisiae for terpenoids biosynthesis].

Terpenoids are a group of structurally diverse compounds with good biological activities and versatile functions such as anti-cancer and immunity-enhancing effects, and are widely used in food, healthcare and medical industries. Facilitated by the increasing understandings on the natural biosynthetic pathways of terpenoids in recent years, Saccharomyces cerevisiae has been engineered into high-yield strains for production of a variety of terpenoids, some of which have reached or become close to the level required by industrial production. In this connection, synthetic biology driven biotechnological production of terpenoids has become a promising alternative to chemical synthesis and traditional extraction approaches. This article summarizes the recent process in engineering S. cerevisiae for terpenoids biosynthesis, highlighting the effect of synthetic biology strategies by taking a couple of typical terpenoids as examples.