Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.     

Cover Image,Volume 116, Number 6, June 2019

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl₃ and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.     

Engineering yeast subcellular compartments for increased production of the lipophilic natural products ginsenosides

Eukaryotic yeasts have a variety of subcellular compartments and are ideal platform strains for the construction of complex heterologous natural product biosynthesis pathways. Improving the synthesis efficiency of microbial cell factories through the utilization and modification of subcellular compartments by synthetic biology has good application prospects. Here, we used the yeast PLN1 protein to target the normally endoplasmic reticulum (ER)-localized cytochrome P450 enzyme protopanaxadiol (PPD) synthase (PPDS) to lipid droplets (LDs), which are the storage organelles of the PPDS substrate dammarenediol-II (DD). The efficiency of converting DD to PPD was significantly increased by 394%, and the conversion rate of DD increased from 17.4% to 86.0%. Furthermore, increasing the volume of LDs can significantly enhance the production of DD and its derivatives, but the change in the ratio of the volume and surface area of LDs decreased the conversion efficiency of DD to PPD. Additionally, the biosynthetic pathways of the PPD-type saponin ginsenoside compound K (CK) was reconstituted in a PPD-producing chassis strain, and CK production reached 21.8 mg/L/OD, 4.4-fold higher compared to the native ER-expression strategy. Next, we enhanced the expression of the Pn3-29 gene module to further reduce the accumulation of PPD and increase the production of CK to 41.3 mg/L/OD. Finally, the CK titer of the resulting strain reached 5 g/L in 5 L fed-batch fermentations. This study provides a new strategy for engineering yeast to produce complex natural products.     

CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli

Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5 g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40 g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these variants, we constructed a combinatorial library containing 256 total variants from four different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1 g/L at 24 h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62 g/L/h (which was 0.22 g/L/h above the parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.     

Biosynthesis of natural flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.     

Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration

NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, is crucial to maximizing production titers. We have rationally engineered a synthetic version of the Entner–Doudoroff pathway from Zymomonas mobilis that increased the NADPH regeneration rate in Escherichia coli MG1655 by 25-fold. To do this, we combined systematic design rules, biophysical models, and computational optimization to design synthetic bacterial operons expressing the 5-enzyme pathway, while eliminating undesired genetic elements for maximum expression control. NADPH regeneration rates from genome-integrated pathways were estimated using a NADPH-binding fluorescent reporter and by the productivity of a NADPH-dependent terpenoid biosynthesis pathway. We designed and constructed improved pathway variants by employing the RBS Library Calculator to efficiently search the 5-dimensional enzyme expression space and by performing 40 cycles of MAGE for site-directed genome mutagenesis. 624 pathway variants were screened using a NADPH-dependent blue fluorescent protein, and 22 were further characterized to determine the relationship between enzyme expression levels and NADPH regeneration rates. The best variant exhibited 25-fold higher normalized mBFP levels when compared to wild-type strain. Combining the synthetic Entner–Doudoroff pathway with an optimized terpenoid pathway further increased the terpenoid titer by 97%.     

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The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3′,5′-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP–cyclic adenosine 3′,5′-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP–CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.     

The synthetic integron: an in vivo genetic shuffling device

As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with ‘regulatory’ elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.     

Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags

Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.