Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus

Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus. It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics.     

Titer improvement of iso-migrastatin in selected heterologous <Emphasis Type="Italic">Streptomyces</Emphasis> hosts and related analysis of mRNA expression by quantitative RT–PCR

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO₃ to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.     

Engineering Saccharomyces cerevisiae for high yield production of α-amyrin via synergistic remodeling of α-amyrin synthase and expanding the storage pool

α-Amyrin is a plant-originated high-valued triterpene that is highly effective against several pathological ailments. α-Amyrin production by engineered Saccharomyces cerevisiae has been achieved by introducing α-amyrin synthase (αAS). However, the low yield of α-amyrin highly limits its industrial application; the low catalytic activity of αAS and the toxic effect of α-amyrin have been considered key elements. In this study, the highest yield of α-amyrin was obtained in engineered S. cerevisiae by remodeling α-amyrin synthase MdOSC1 and expanding the storage pool. The yield of α-amyrin was increased to 11-fold higher than that of the control by the triple mutant MdOSC1^{N11T/P250H/P373A} obtained based on the modeling analysis. Furthermore, key genes of MVA pathway were overexpressed to provide sufficient precursors, and DGA1 (Diacylglycerol acyltransferase) was overexpressed to expand the intracellular storage capacity. Finally, the as-constructed aAM12 strain produced 213.7 ± 12.4 mg/L α-amyrin in the shake flask and 1107.9 ± 76.8 mg/L in fed-batch fermentation; the fermentation yield was 106-fold higher than that of the original aAM1 strain under the same conditions, representing the highest α-amyrin yield in yeast reported to date. Microbial production of α-amyrin with over 1 g/L will be suitable for commercialization and can accelerate the industrial production of α-amyrin in yeast.     

Engineering Saccharomyces cerevisiae for isoprenol production

Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.     

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.     

Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFP0) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS115/pPICZαA/rhAFP0, which produces unglycosylated rhAFP0 and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.     

5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway

ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C₅ pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2 g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1 g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering.     

Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of (+)-valencene and its chemical conversion to (+)-nootkatone

(+)-Nootkatone is a valuable, functional sesquiterpene that is widely used in food, cosmetics, pharmaceutical, agriculture, and other fields. However, only traces of it accumulate in plants, which is insufficient to meet the market demand. Therefore, commercial (+)-nootkatone is currently synthesized from (+)-valencene. Here, we engineered Saccharomyces cerevisiae to achieve high production of (+)-valencene. Employing gene screening, protein engineering and biosynthetic pathway optimization, we achieved 12.4 g/L (+)-valencene production with the mutant strain. This titer was further increased to 16.6 g/L, the highest titer reported to date, by coupling critical factors for cell growth and biochemical pathway induction. Subsequently, (+)-nootkatone was chemically synthesized from bio-fermented (+)-valencene with a yield of 80%. This study achieved efficient microbial synthesis of (+)-valencene, which may be utilized in industrial production and stabilize the supply of (+)-nootkatone.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Metabolic engineering of E. coli for β-alanine production using a multi-biosensor enabled approach

β-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of β-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo β-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in β-alanine yield (0.85 mol β-alanine/mole glucose) and specific β-alanine production (0.74 g/L/OD₆₀₀), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol β-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L β-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of β-alanine.     

Studies on process optimization methods for rapamycin production using Streptomyces hygroscopicus ATCC 29253

Rapamycin is a high-value product finding immense use as a drug, in organ transplantation, and as a potential immunosuppressant. Optimization of fermentation parameters of rapamycin production by Streptomyces hygroscopicus NRRL 5491 has been carried out. The low titer value of rapamycin in the original producer strain limits its applicability at industrial level. This study aims at improving the production of rapamycin by optimizing the nutrient requirements. Addition of l-lysine increased the production of rapamycin up to a significant level which supports the fact that it acts as precursor for rapamycin production, as found in previous studies. Effect of optimized medium on the Streptomyces growth rate as well as rapamycin production has been studied. The optimization study incorporates one at a time parameter optimization studies followed by tool-based hybrid methodology. This methodology includes the Plackett–Burman design (PBD) method, artificial neural networks (ANN), and genetic algorithms (GA). PBD screened mannose, soyabean meal, and l-lysine concentrations as significant factors for rapamycin production. ANN was used to construct rapamycin production model. This strategy has led to a significant increase of rapamycin production up to 320.89 mg/L at GA optimized concentrations of 25.47, 15.39, and 17.48 g/L for mannose, soyabean meal, and l-lysine, respectively. The present study must find its application in scale-up study for industrial level production of rapamycin.     

A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20^F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.     

Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering

With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Δpgi, Δppc and ΔpldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control.     

Engineering Escherichia coli to convert acetic acid to free fatty acids

Fatty acids (FAs) are promising precursors of advanced biofuels. This study investigated conversion of acetic acid (HAc) to FAs by an engineered Escherichia coli strain. We combined established genetic engineering strategies including overexpression of acs and tesA genes, and knockout of fadE in E. coli BL21, resulting in the production of ~1 g/L FAs from acetic acid. The microbial conversion of HAc to FAs was achieved with ~20% of the theoretical yield. We cultured the engineered strain with HAc-rich liquid wastes, which yielded ~0.43 g/L FAs using waste streams from dilute acid hydrolysis of lignocellulosic biomass and ~0.17 g/L FAs using effluent from anaerobic-digested sewage sludge. ¹³C-isotopic experiments showed that the metabolism in our engineered strain had high carbon fluxes toward FAs synthesis and TCA cycle in a complex HAc medium. This proof-of-concept work demonstrates the possibility for coupling the waste treatment with the biosynthesis of advanced biofuel via genetically engineered microbial species.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.     

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of β-alanine

β-Alanine is an important β-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of β-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of β-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of β-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient β-alanine biosynthesis was used to construct a β-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative β-alanine exporter was newly identified, and its overexpression further improved the β-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of β-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.     

Production of chromopyrrolic acid by coexpression of inkOD in a heterologous host Streptomyces albus

Two ink genes, inkO and inkD, responsible for the earliest steps of K252a biosynthetic pathway, from Nonomurea longicantena JCM 11136 were heterologously coexpressed in Streptomyces albus J1074. The resultant strain accumulated compound that was purified by HPLC and studied by NMR. Coexpression of inkOD yielded chromopyrrolic acid, the key intermediate in an indolocarbazole biosynthesis.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.