Modular Pathway Engineering Research Articles

Systematic metabolic engineering of Yarrowia lipolytica for the enhanced production of erythritol

In recent times, there has been a growing interest in exploring microbial strains that exhibit enhanced erythritol productivity. Nonetheless, the lack of advanced synthetic biology tools has limited rapid strain development. In this study, the CRISPR/Cas9 system was employed to genetically modify Yarrowia lipolytica at the chromosomal level, which could improve the production of erythritol while saving the time required to markers recovery, and realizing the rapid construction of high-erythritol strains. Firstly, the basic strain E004 was generated by increasing the efficiency of homologous recombination and regulating the erythritol degradation pathway. Secondly, eleven key gene targets and a strong promoter 8UAS1BXPR2-PTEFin was obtained by target screening and promoter engineering. Finally, based on modular pathway engineering and morphological engineering, the high production of erythritol was achieved successfully. The best-engineered strain E326 produced 256 g/L erythritol in a 5-L bioreactor, which is the highest production level reported so far in Y. lipolytica.

Efficient Production of Chlorogenic Acid in Escherichia coli Via Modular Pathway and Cofactor Engineering.

Chlorogenic acid is a natural phenolic compound widely used in the food and daily chemical industries. Compared to plant extraction, microbial cell factories provide a green and sustainable production method for the production of chlorogenic acid. However, complex metabolic flux distribution and potential byproducts limited the biosynthesis of chlorogenic acid in microorganisms. A de novo biosynthesis pathway for chlorogenic acid was constructed in Escherichia coli via modular engineering. Increasing the shikimate pathway flux greatly promoted chlorogenic acid production, and the influence of pyruvate metabolism on chlorogenic acid synthesis was also explored. The supply of cofactors for the key enzymes quinate/shikimate 5-dehydrogenase (YdiB) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC) was enhanced by a cofactor regeneration system. Furthermore, mutants of YdiB were verified for chlorogenic acid production in vivo. Chlorogenic acid browning occurred when the buffer pH of the buffer exceeded 6.0, but two-stage pH control achieved a chlorogenic acid titer of 2789.2 mg/L in a 5 L fermenter, the highest reported to date. This study provided a strategy for the efficient production of chlorogenic acid from simple carbon sources.

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

Metabolic Engineering of De Novo Pathway for the Production of 2'-Fucosyllactose in Escherichia coli.

2'-Fucosyllactose (2'-FL), one of the most abundant oligosaccharides in human milk, has gained increased attention owing to its nutraceutical and pharmaceutical potential. However, limited availability and high-cost of preparation have limited its widespread application and in-depth investigation of its potential functions. Here, a modular pathway engineering was implemented to construct an Escherichia coli strain to improve the biosynthesis titer of 2'-FL. Before overexpression of manB, manC, gmd, wcaG, and heterologous expression of futC, genes wcaJ and lacZ encoding UDP-glucose lipid carrier transferase and β-galactosidase, respectively, were inactivated from E. coli BL21 (DE3) with the CRISPR-Cas9 system, which inhibited the production of 2'-FL. The results showed that final shake flask culture yielded a 3.8-fold increase in 2'-FL (0.98g/L) from the engineered strain ELC07. Fed-batch fermentation conditions were optimized in a 3-L bioreactor. The highest titer of 2'-FL (18.22g/L) was obtained, corresponding to a yield of 0.25g/g glycerol and a substrate conversion of 0.88g/g lactose.

Large-scale bioproduction of natural astaxanthin in Yarrowia lipolytica

Astaxanthin is a high-value chemical with strong antioxidant, anti-inflammatory and coloration properties. Here, a set of novel genetic engineering and culturing strategies for efficient astaxanthin bioproduction in Yarrowia lipolytica is reported. Modular pathway engineering and the fusion of two key enzymes (geranylgeranyl pyrophosphate synthase and bifunctional lycopene cyclase/phytoene synthase) yielded a recombinant strain with enhanced β-carotene and astaxanthin production, accumulating astaxanthin up to 131.9 mg/L in small-scale cultures with sucrose and 587.3 mg/L in a 3-L bioreactor with glucose as carbon sources, respectively. Notably, formation of intracellular carotenoid crystals was observed. Addition of oil overlayer at different regimes in 3-L bioreactors promoted secretion of astaxanthin and increased astaxanthin titer up to 973.4 g/L. Lastly, fed-batch fermentation in a 100-L bioreactor with glycerol as the carbon source resulted in a final astaxanthin titer of 812.3 mg/L after 114 h, representing the highest astaxanthin titer reported so far in large-scale cultures.

Enhanced biosynthesis of d-tagatose from maltodextrin through modular pathway engineering of recombinant Escherichia coli

In our previous study, a whole-cell biocatalytic system involving α-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate isomerase, tagatose 1,6-bisphosphate aldolase, and phosphoglycolate phosphatase, was constructed to produce d-tagatose from maltodextrin. However, the biotransformation efficiency of this whole-cell catalyst was low because of the unbalanced ratio of each enzyme and metabolic flux of the intermediates. Therefore, in this study, the biosynthesis of d-tagatose was enhanced by optimizing vectors and improving the expression of rate-limiting enzymes through the construction of multi-copy genes to regulate expression levels. The conversion rate increased from 20.8% after 24 h to 25.2% after 3 h using 10 g/L maltodextrin as the substrate. Furthermore, the genes in the bypass pathways in Escherichia coli, pfka and zwf, were deleted to increase the accumulation of intermediates. The strain ER-2GatZ (ΔpΔz) produced 3.383 g/L d-tagatose using 10 g/L substrate after 3 h, which was 1.34 times that of the wild strain. This work exemplifies the use of modular engineering to enhance d-tagatose production using whole-cell catalysts.

Metabolic engineering strategies of de novo pathway for enhancing 2'-fucosyllactose synthesis in Escherichia coli.

Summary2′‐Fucosyllactose (2′‐FL), one of the most abundant human milk oligosaccharides (HMOs), is used as a promising infant formula ingredient owing to its multiple health benefits for newborns. However, limited availability and high‐cost preparation have restricted its extensive use and intensive research on its potential functions. In this work, a powerful Escherichia coli cell factory was developed to ulteriorly increase 2′‐FL production. Initially, a modular pathway engineering was strengthened to balance the synthesis pathway through different plasmid combinations with a resulting maximum 2′‐FL titre of 1.45 g l−1. To further facilitate the metabolic flux from GDP‐l‐fucose towards 2′‐FL, the CRISPR‐Cas9 system was utilized to inactivate the genes including lacZ and wcaJ, increasing the titre by 6.59‐fold. Notably, the co‐introduction of NADPH and GTP regeneration pathways was confirmed to be more conducive to 2′‐FL formation, achieving a 2′‐FL titre of 2.24 g l−1. Moreover, comparisons of various exogenous α1,2‐fucosyltransferase candidates revealed that futC from Helicobacter pylori generated the highest titre of 2′‐FL. Finally, the viability of scaled‐up production of 2′‐FL was evidenced in a 3 l bioreactor with a maximum titre of 22.3 g l−1 2′‐FL and a yield of 0.53 mole 2′‐FL mole−1 lactose.

Reprogramming Escherichia coli Metabolism for Bioplastics Synthesis from Waste Cooking Oil.

The recycle and reutilization of food wastes is a promising alternative for supporting and facilitating circular economy. However, engineering industrially relevant model organisms to use food wastes as their sole carbon source has remained an outstanding challenge so far. Here, we reprogrammed Escherichia coli metabolism using modular pathway engineering followed by laboratory adaptive evolution to establish a strain that can efficiently utilize waste cooking oil (WCO) as the sole carbon source to produce monomers of bioplastics, namely, medium-chain α,ω-dicarboxylic acids (MCDCAs). First, the biosynthetic pathway of MCDCAs was designed and rewired by modifying the β-oxidation pathway and introducing an ω-oxidation pathway. Then, metabolic engineering and laboratory adaptive evolution were applied for improving the pathway efficiency of fatty acids utilization. Finally, the engineered strain E.coli AA0306 was able to produce 15.26 g/L MCDCAs with WCO as the sole carbon source. This study provides an economically attractive strategy for biomanufacturing bioplastics from food wastes, which has a great potentiality to be developed as a wide range of enabling biotechnologies for achieving green revolution.

Combinatorial Modular Pathway Engineering for Guanosine 5'-Diphosphate-l-fucose Production in Recombinant Escherichia coli.

Guanosine 5'-diphosphate (GDP)-l-fucose is an important nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. Here, a combinatorial modular pathway engineering strategy was implemented to efficiently increase the intracellular titers of GDP-l-fucose in engineered Escherichia coli. The de novo GDP-l-fucose synthesis pathway was partitioned into two modules and fine-tuned at both transcriptional and translational levels, which remarkably improved the GDP-l-fucose production. In addition, the gene encoding the UDP-glucose lipid carrier transferase (WcaJ) was inactivated to eliminate the competing metabolite pathway from GDP-l-fucose to colanic acid. Furthermore, cofactors were regenerated to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve a titer of 18.33 mg/L in shake-flask cultivation, showed 106.21 mg/L intracellular GDP-l-fucose accumulation and a DCW-specific GDP-l-fucose content of 4.28 mg/g through fed-batch cultivation. In general, this study demonstrated that the utilization of combinatorial modular pathway engineering significantly improved the de novo synthesis of GDP-l-fucose in engineered E. coli.

A modular pathway engineering strategy for the high-level production of \u03b2-ionone in Yarrowia lipolytica

BackgroundThe GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance.ResultsIn this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation.ConclusionsAn efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.

De novo Biosynthesis of Odd-Chain Fatty Acids in Yarrowia lipolytica Enabled by Modular Pathway Engineering.

Microbial oils are regarded as promising alternatives to fossil fuels as concerns over environmental issues and energy production systems continue to mount. Odd-chain fatty acids (FAs) are a type of valuable lipid with various applications: they can serve as biomarkers, intermediates in the production of flavor and fragrance compounds, fuels, and plasticizers. Microorganisms naturally produce FAs, but such FAs are primarily even-chain; only negligible amounts of odd-chain FAs are generated. As a result, studies using microorganisms to produce odd-chain FAs have had limited success. Here, our objective was to biosynthesize odd-chain FAs de novo in Yarrowia lipolytica using inexpensive carbon sources, namely glucose, without any propionate supplementation. To achieve this goal, we constructed a modular metabolic pathway containing seven genes. In the engineered strain expressing this pathway, the percentage of odd-chain FAs out of total FAs was higher than in the control strain (3.86 vs. 0.84%). When this pathway was transferred into an obese strain, which had been engineered to accumulate large amounts of lipids, odd-chain fatty acid production was 7.2 times greater than in the control (0.05 vs. 0.36 g/L). This study shows that metabolic engineering research is making progress toward obtaining efficient cell factories that produce odd-chain FAs.

Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis

BackgroundLacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation.ResultsIn this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy.ConclusionsIn general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.

Eudesmane-type sesquiterpene diols directly synthesized by a sesquiterpene cyclase in Tripterygium wilfordii.

Cryptomeridiol, a typical eudesmane diol, is the active principle component of the antispasmodic Proximol. Although it has been used for many years, the biosynthesis pathway of cryptomeridiol has remained blur. Among terpenoid natural products, terpenoid cyclases are responsible for cyclization and generation of hydrocarbon backbones. The cyclization is mediated by carbocationic cascades and ultimately terminated via deprotonation or nucleophilic capture. Isoprene precursors are, respectively, converted into hydrocarbons or hydroxylated backbones. A sesquiterpene cyclase in Tripterygium wilfordii (TwCS) was determined to directly catalyze (E,E)-farnesyl pyrophosphate (FPP) to unexpected eudesmane diols, primarily cryptomeridiol. The function of TwCS was characterized by a modular pathway engineering system in Saccharomyces cerevisiae The major product determined by NMR spectroscopy turned out to be cryptomeridiol. This unprecedented production was further investigated in vitro, which verified that TwCS can directly produce eudesmane diols from FPP. Some key residues for TwCS catalysis were screened depending on the molecular model of TwCS and mutagenesis studies. As cryptomeridiol showed a small amount of volatile and medicinal properties, the biosynthesis of cryptomeridiol was reconstructed in S. cerevisiae Optimized assays including modular pathway engineering and the CRISPR-cas9 system were successfully used to improve the yield of cryptomeridiol in the S. cerevisiae The best engineered strain TE9 (BY4741 erg9::Δ-200-176 rox1::mut/pYX212-IDI + TwCS/p424-tHMG1) ultimately produced 19.73 mg/l cryptomeridiol in a shake flask culture.

Modular pathway engineering for resveratrol and piceatannol production in engineered Escherichia coli.

Resveratrol and its ortho-hydroxylated derivative piceatannol were biosynthesized by modular pathway engineering in Escherichia coli. The biosynthetic pathway was divided into three different modules. Module I includes polyketide biosynthetic genes; module II genes include acetyl-CoA and malonyl-CoA pool-enhancing genes from three different organisms; and module III genes are regiospecific 3'-hydroxylating enzymes. E. coli BL21(DE3) with module I produced 8.6mg/L of resveratrol from exogenously fed 1mM p-coumaric acidafter 72 h. Combination of module I and acetyl-CoA supplementing module IIb genes from N. farcinica IFM10152 produced 2.5-fold higher (60mg/L) titer of resveratrol than the module IIa genes from E. coli. The exogenous supplementation of sodium acetate further enhanced production to 64mg/L. Furthermore, module I with module IIc harboring matBC from S. coelicolor A3(2) produced 73mg/L of resveratrol, which was elevated to 151mg/L upon supplementing disodium malonate exogenously. This increment is 17.5-fold higher than module I harboring E. coli BL21(DE3). The combination of module I and two different module II genes yielded 137mg/L resveratrol when supplemented with both sodium acetate and disodium malonate. The high resveratrol-producing combination module was further modified with incorporation of hpaBC for the ortho-hydroxylation of resveratrol to produce piceatannol. The engineered strain harboring modules I, IIc and III produced 124mg/L of piceatannol, the highest titer after 72h in disodium malonate-supplemented strain, which is 2-fold higher than in non-supplemented strain. The remaining resveratrol was about 30mg/L. Furthermore, caffeic acid (85.5mg/L) was also produced in the same strain. Resveratrol and piceatannol were biosynthesized along with caffeic acid by three different modules overexpressing acetate and malonate assimilation pathway genes from three different sources. The production titer of both resveratrol and piceatannol could be achieved higher upon blocking acetyl-CoA and malonyl-CoA utilizing pathway genes in host strain.

Modular pathway engineering of key carbon-precursor supply-pathways for improved N-acetylneuraminic acid production in Bacillus subtilis.

N-acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non-ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors-N-acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)-and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc-producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine-6-phosphate acetyl-transferase (GNA1), N-acetylglucosamine 2-epimerase (AGE), and N-acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0-fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner-Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32-fold and 2.64-fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose-malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.

Rapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression

Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering. Along the way, combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits. In this report, we present an efficient evolutionary approach to build a range of regulatory control elements. The reported method allows for rapid construction of promoter, 5′UTR, terminator and trans-activating RNA libraries. Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter. This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix. Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics. Specifically, both the promoter library and 5′UTR library exhibits gene expression dynamics spanning across three order of magnitude. The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern. The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements. These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization.

Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering.

Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc.

Stepwise modular pathway engineering of Escherichia coli for efficient one-step production of (2S)-pinocembrin

Developing efficient microbial processes to produce flavonoids has been a metabolic engineering goal over the past decade due to their important functions. Previously, the de novo production of the main flavonoid precursor (2S)-pinocembrin was achieved. However, low productivity and two separate fermentation steps made it inappropriate for industrial scale low-cost production. Here, a stepwise modular engineering approach was introduced to systematically identify and eliminate metabolic pathway bottlenecks. The overall pathway was firstly divided into four modules and analysis then revealed that efficient conversion of l-phenylalanine to (2S)-pinocembrin is the major limiting factor. Therefore, the pathway from l-phenylalanine to (2S)-pinocembrin was re-cast into three modules to alleviate this bottleneck by modifying both the gene copy number and promoter strength. Furthermore, the expression of the rate-limiting enzyme PAL was found to be correlated with 5′ region of mRNA structure. The efficiency of the synthetic pathway was then improved by customizing the PAL expression level based on modification of the mRNA secondary structure. Fed-batch cultivation of engineered strains in a 3-L fermentor resulted in a final (2S)-pinocembrin production of 432.4mg/L. The results presented here pave the way for the development of an economical and simple process for microbial production of flavonoids.

Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering

A Bacillus amyloliquefaciens strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the epsA-O (responsible for extracellular polysaccharide synthesis), sac (responsible for levan synthesis), lps (responsible for lipopolysaccharide synthesis) and pta (encoding phosphotransacetylase) genes, showed increased γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15g/L. The γ-PGA degrading genes pgdS (encoding poly-gamma-glutamate depolymerase) and cwlO (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased γ-PGA production from 4.15 to 9.18g/L. The autoinducer AI-2 synthetase gene luxS was deleted in NK-E10 strain and the resulting NK-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54g/L). In addition, we overexpressed the pgsBCA genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-PGA production. Finally, the rocG gene (encoding glutamate dehydrogenase) and the glnA gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The rocG-repressed NK-anti-rocG strain exhibited the highest γ-PGA titer (11.04g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final γ-PGA titer of 20.3g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced γ-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are also useful for engineering cell factories for the production of γ-PGA or of other valuable metabolites.