Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use ...of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli . This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.
In this work, Liu et al. used metabolic engineering strategies to construct S. cerevisiae strains for high‐level production of tyrosol and salidroside from glucose. Finally, titers of 9.90 ± 0.06 g ...l−1 of tyrosol and 26.55 ± 0.43 g l−1 of salidroside were achieved in 5 l bioreactors, both are the highest titers reported to date.
Summary
Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high‐level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose‐5‐phosphate ketol‐isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l−1 in shake flasks, which were approximately 26‐fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l−1 were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l−1 of tyrosol and 26.55 ± 0.43 g l−1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value‐added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.
The hyphenation of ion mobility spectrometry with high-resolution mass spectrometry has been widely used in the characterization of various metabolites. Nevertheless, such a powerful tool remains ...largely unexplored in natural products research, possibly mainly due to the lack of available compounds. To evaluate the ability of collision cross-sections (CCSs) in characterizing compounds, especially isomeric natural products, here we measured and compared the traveling-wave IMS-derived nitrogen CCS values for 75 marine-derived aphidicolanes. We established a CCS database for these compounds which contained 227 CCS values of different adducts. When comparing the CCS differences, 36 of 57 pairs (over 60%) of chromatographically neighboring compounds showed a ΔCCS over 2%. What is more, 64 of 104 isomeric pairs (over 60%) of aphidicolanes can be distinguished by their CCS values, and 13 of 18 pairs (over 70%) of chromatographically indistinguishable isomers can be differentiated from the mobility dimension. Our results strongly supported CCS as an important parameter with good orthogonality and complementarity with retention time. CCS is expected to play an important role in distinguishing complex and diverse marine natural products.
Celastrus angulatus Maxim is a kind of crucial and traditional insecticidal plant widely distributed in the mountains of southwest China. Celangulin V is the efficient insecticidal sesquiterpenoid of ...C. angulatus and widely used in pest control in China, but the low yield and discontinuous supply impeded its further popularization and application. Fortunately, the development of synthetic biology provided an opportunity for sustainable supply of Celangulin V, for which understanding its biosynthetic pathway is indispensable.
In this study, six cDNA libraries were prepared from leaf and root of C. angulatus before global transcriptome analyses using the BGISEQ-500 platform. A total of 104,950 unigenes were finally obtained with an average length of 1200 bp in six transcriptome databases of C. angulatus, in which 51,817 unigenes classified into 25 KOG classifications, 39,866 unigenes categorized into 55 GO functional groups, and 48,810 unigenes assigned to 135 KEGG pathways, 145 of which were putative biosynthetic genes of sesquiterpenoid and triterpenoid. 16 unigenes were speculated to be related to Celangulin V biosynthesis. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis.
This study is the first report on transcriptome analysis of C. angulatus, and 16 unigenes probably involved in the biosynthesis of Celangulin V were finally collected. The transcriptome data will make great contributions to research for this specific insecticidal plant and the further gene mining for biosynthesis of Celangulin V and other sesquiterpene polyol esters.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Due to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential ...applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.
By introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.
Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.
Metabolic engineering strategies for terpenoid production have mainly focused on bottlenecks in the supply of precursor molecules and cytotoxicity to terpenoids. In recent years, the strategies ...involving compartmentalization in eukaryotic cells has rapidly developed and have provided several advantages in the supply of precursors, cofactors and a suitable physiochemical environment for product storage. In this review, we provide a comprehensive analysis of organelle compartmentalization for terpenoid production, which can guide the rewiring of subcellular metabolism to make full use of precursors, reduce metabolite toxicity, as well as provide suitable storage capacity and environment. Additionally, the strategies that can enhance the efficiency of a relocated pathway by increasing the number and size of organelles, expanding the cell membrane and targeting metabolic pathways in several organelles are also discussed. Finally, the challenges and future perspectives of this approach for the terpenoid biosynthesis are also discussed.
Synthetic genomes were designed based on an understanding of natural genomic information, offering an opportunity to engineer and investigate biological systems on a genome-wide scale. Currently, the ...designer version of the
genome and the
genome, as well as most of the
genome, have been synthesized, and through the cycles of design-build-test and the following engineering of synthetic genomes, many fundamental questions of genome biology have been investigated. In this review, we summarize the use of synthetic genome engineering to explore the structure and function of genomes, and highlight the unique values of synthetic genomics.
Taxadiene is an important precursor in taxol biosynthesis pathway, but its biosynthesis in eukaryotic cell factories is limited, which seriously hinders the biosynthesis of taxol. In this study, it ...is found that there was the catalysis compartmentalization between two key exogenous enzymes of geranylgeranyl pyrophosphate synthase and taxadiene synthase (TS) for taxadiene synthesis progress, due to their different subcellular localization. Firstly, the enzyme-catalysis compartmentalization was overcome by means of the intracellular relocation strategies of taxadiene synthase, including N-terminal truncation of taxadiene synthase and enzyme fusion of GGPPS-TS. With the help of two strategies for enzyme relocation, the taxadiene yield was increased by 21% and 54% respectively, among them the GGPPS-TS fusion enzyme is more effective. Further, the expression of GGPPS-TS fusion enzyme was improved
the multi-copy plasmid, resulting that the taxadiene titer was increased by 38% to 21.8 mg/L at shake-flask level. Finally, the maximum taxadiene titer of 184.2 mg/L was achieved by optimization of the fed-batch fermentation conditions in 3 L bioreactor, which is the highest reported titer of taxadiene biosynthesis accomplished in eukaryotic microbes. This study provides a successful example for improving biosynthesis of complex natural products by solving the critical problem of multistep enzymes catalysis compartmentalization.
21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone ...(17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity.
In this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known.
Heterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.
The conversion of
β
-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by
β
-carotene hydroxylase (CrtZ) and
β
...-carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in
Saccharomyces cerevisiae
by introducing heterologous CrtZ and CrtW into an existing high
β
-carotene producing strain. Both genes
crtZ
and
crtW
were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from
Brevundimonas vesicularis
DC263 and CrtZ from
Alcaligenes
sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of
crtZ
in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on
crtZ
transcription level and 39.1-fold enhancement on the transcriptional ratio of
crtZ
to
crtW
were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of
crtZ
to
crtW
were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both
crtZ
and
crtW
were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes.
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