Toward Photosynthetic Mammalian Cells through Artificial Endosymbiosis.

Photosynthesis in plants occurs within specialized organelles known as chloroplasts, which are postulated to have originated through endosymbiosis with cyanobacteria. In nature, instances are also observed wherein specific invertebrates engage in symbiotic relationships with photosynthetic bacteria, allowing them to subsist as photoautotrophic organisms over extended durations. Consequently, the concept of engineering artificial endosymbiosis between mammalian cells and cyanobacteria represents a promising avenue for enabling photosynthesis in mammals. The study embarked with the identification of Synechocystis PCC 6803 as a suitable candidate for establishing a long-term endosymbiotic relationship with macrophages. The cyanobacteria internalized by macrophages exhibited the capacity to rescue ATP deficiencies within their host cells under conditions of illumination. Following this discovery, a membrane-coating strategy is developed for the intracellular delivery of cyanobacteria into non-macrophage mammalian cells. This pioneering technique led to the identification of human embryonic kidney cells HEK293 as optimal hosts for achieving sustained endosymbiosis with Synechocystis PCC 6803. The study offers valuable insights that may serve as a reference for the eventual achievement of artificial photosynthesis in mammals.

Efficient production of protocatechuic acid using systems engineering of Escherichia coli.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in Escherichia coli. In this study, a high-level strain of 3-dehydroshikimate, E. coli DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain E. coli SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity ApAroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from Acinetobacter pittii PHEA-2 (ApAroZ) were clarified. The variant ApAroZR363A, achieved by modulating the conformational dynamics of ApAroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain E. coli PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

Systems engineering Escherichia coli for efficient production p-coumaric acid from glucose.

P-coumaric acid (p-CA), a pant metabolite with antioxidant and anti-inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p-CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l-phenylalanine-producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p-CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N-terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3-fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9-folds, 1.3-folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p-CA titer, respectively. These optimizations significantly enhance p-CA production, in a 5-L fermenter using fed-batch fermentation, the p-CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

Engineering microbial cell viability for enhancing chemical production by second codon engineering.

Microbial cell factories offer a promising strategy for the sustainable production of industrial chemicals from renewable biomass feedstock. However, their performance is often limited by poor microbial cell viability (MCV). Here, MCV was engineered to enhance chemical production by optimizing the regulation of lifespan-specific genes to reduce the accumulation of reactive oxygen species (ROS). In Escherichia coli, MCV was improved by reducing ROS accumulation using second codon engineering to regulate hypoxia-inducible transcription factor (arcA), resulting in lysine production up to 213 g L-1 with its productivity 5.90 g L-1·h-1. In Saccharomyces cerevisiae, MCV was increased by decreasing ROS accumulation using second codon engineering to fine-tune ceramide synthase (lag1), leading to glucaric acid production up to 9.50 g L-1 with its productivity 0.057 g L-1·h-1. These results demonstrate that engineering MCV is a potential strategy to boost the performance of microbial cell factories in industrial processes.

Enhancing tryptophan production by balancing precursors in Escherichia coli.

Tryptophan, an essential aromatic amino acid, is widely used in animal feed, food additives, and pharmaceuticals. Although sustainable and environmentally friendly, microbial tryptophan production from renewable feedstocks is limited by low biosynthesis and transport rates. Here, an Escherichia coli strain capable of efficient tryptophan production was generated by improving and balancing the supply of precursors and by engineering membrane transporters. Tryptophan biosynthesis was increased by eliminating negative regulatory factors, blocking competing pathways, and preventing tryptophan degradation. Promoter engineering balanced the supply of the precursors erythrose-4-phosphate and phosphoenolpyruvate, as well as the availability of serine. Finally, the engineering of tryptophan transporters prevented feedback inhibition and growth toxicity. Fed-batch fermentation of the final strain (TRP12) in a 5 L bioreactor produced 52.1 g·L-1 of tryptophan, with a yield of 0.171 g·g-1 glucose and productivity of 1.45 g·L-1 ·h-1 . The metabolic engineering strategy described here paves the way for high-performance microbial cell factories aimed at the production of tryptophan as well as other valuable chemicals.

Engineering membrane asymmetry to increase medium-chain fatty acid tolerance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an attractive chassis for the production of medium-chain fatty acids, but the toxic effect of these compounds often prevents further improvements in titer, yield, and productivity. To address this issue, Lem3 and Sfk1 were identified from adaptive laboratory evolution mutant strains as membrane asymmetry regulators. Co-overexpression of Lem3 and Sfk1 [Lem3(M)-Sfk1(H) strain] through promoter engineering remodeled the membrane phospholipid distribution, leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane. As a result, membrane potential and integrity were increased by 131.5% and 29.2%, respectively; meanwhile, the final OD600 in the presence of hexanoic acid, octanoic acid, and decanoic acid was improved by 79.6%, 73.4%, and 57.7%, respectively. In summary, this study shows that membrane asymmetry engineering offers an efficient strategy to enhance medium-chain fatty acids tolerance in S. cerevisiae, thus generating a robust industrial strain for producing high-value biofuels.

Production of phenylpyruvic acid by engineered L-amino acid deaminase from Proteus mirabilis.

OBJECTIVES: This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). RESULTS: L-amino acid deaminase from Proteus mirabilis was expressed in Escherichia coli BL21 (DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145/L341) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. CONCLUSIONS: The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.

Dynamic regulation of membrane integrity to enhance l-malate stress tolerance in Candida glabrata.

Microbial cell factories provide a sustainable and economical way to produce chemicals from renewable feedstocks. However, the accumulation of targeted chemicals can reduce the robustness of the industrial strains and affect the production performance. Here, the physiological functions of Mediator tail subunit CgMed16 at l-malate stress were investigated. Deletion of CgMed16 decreased the survival, biomass, and half-maximal inhibitory concentration (IC50 ) by 40.4%, 34.0%, and 30.6%, respectively, at 25 g/L l-malate stress. Transcriptome analysis showed that this growth defect was attributable to changes in the expression of genes involved in lipid metabolism. In addition, tolerance transcription factors CgUSV1 and CgYAP3 were found to interact with CgMed16 to regulate sterol biosynthesis and glycerophospholipid metabolism, respectively, ultimately endowing strains with excellent membrane integrity to resist l-malate stress. Furthermore, a dynamic tolerance system (DTS) was constructed based on CgUSV1, CgYAP3, and an l-malate-driven promoter Pcgr-10 to improve the robustness and productive capacity of Candida glabrata. As a result, the biomass, survival, and membrane integrity of C. glabrata 012 (with DTS) increased by 22.6%, 31.3%, and 53.8%, respectively, compared with those of strain 011 (without DTS). Therefore, at shake-flask scale, strain 012 accumulated 35.5 g/L l-malate, and the titer and productivity of l-malate increased by 32.5% and 32.1%, respectively, compared with those of strain 011. This study provides a novel strategy for the rational design and construction of DTS for dynamically enhancing the robustness of industrial strains.

Rational design of a highly efficient catalytic system for the production of PAPS from ATP and its application in the synthesis of chondroitin sulfate.

The compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of adenosine triphosphate (ATP) (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided "ADP expulsion" strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme's flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1 ) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor, and achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. Then, the PAPS catalytic system was combined with the chondroitin 4-sulfotransferase using a one-pot method. Finally, chondroitin sulfate was transformed from chondroitin at a conversion rate of 98.75%. This strategy has great potential for scale biosynthesis of PAPS and chondroitin sulfate.

Dynamic consolidated bioprocessing for direct production of xylonate and shikimate from xylan by Escherichia coli.

Numerous value-added chemicals can be produced using xylan as a feedstock. However, the product yields are limited by low xylan utilization efficiency, as well as by carbon flux competition between biomass production and biosynthesis. Herein, a dynamic consolidated bioprocessing strategy was developed, which coupled xylan utilization and yield optimization modules. Specifically, we achieved the efficient conversion of xylan to valuable chemicals in a fully consolidated manner by optimizing the expression level of xylanases and xylose transporter in the xylan utilization module. Moreover, a cell density-dependent, and Cre-triggered dynamic system that enabled the dynamic decoupling of biosynthesis and biomass production was constructed in the yield optimization module. The final shake flask-scale titers of xylonate, produced through an exogenous pathway, and shikimate, produced through an endogenous pathway, reached 16.85 and 3.2 g L-1, respectively. This study not only provides an efficient microbial platform for the utilization of xylan, but also opens up the possibility for the large-scale production of high value-added chemicals from renewable feedstocks.

Rewiring carbon flux in Escherichia coli using a bifunctional molecular switch.

The unbalanced distribution of carbon flux in microbial cell factories can lead to inefficient production and poor cell growth. Uncoupling cell growth and chemical synthesis can therefore improve microbial cell factory efficiency. Such uncoupling, which requires precise manipulation of carbon fluxes, can be achieved by up-regulating or down-regulating the expression of enzymes of various pathways. In this study, a dynamic turn-off switch (dTFS) and a dynamic turn-on switch (dTNS) were constructed using growth phase-dependent promoters and degrons. By combining the dTFS and dTNS, a bifunctional molecular switch that could orthogonally regulate two target proteins was introduced. This bifunctional molecular switch was used to uncouple cell growth from shikimic acid and D-glucaric acid synthesis, resulting in the production of 14.33 g/L shikimic acid and the highest reported productivity of D-glucaric acid (0.0325 g/L/h) in Escherichia coli MG1655. This proved that the bifunctional molecular switch could rewire carbon fluxes by controlling target protein abundance.

DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals.

Chemical synthesis is a well established route for producing many chemicals on a large scale, but some drawbacks still exist in this process, such as unstable intermediates, multistep reactions, complex process control, etc. Biobased production provides an attractive alternative to these challenges, but how to make cells into efficient factories is challenging. As a key enabling technology to develop efficient cell factories, design-construction-evaluation-optimization (DCEO) biotechnology, which incorporates the concepts and techniques of pathway design, pathway construction, pathway evaluation, and pathway optimization at the systems level, offers a conceptual and technological framework to exploit potential pathways, modify existing pathways and create new pathways for the optimal production of desired chemicals. Here, we summarize recent progress of DCEO biotechnology and examples of its application, and provide insights as to when, what and how different strategies should be taken. In addition, we highlight future perspectives of DCEO biotechnology for the successful establishment of biorefineries.

Engineering synergetic CO2-fixing pathways for malate production.

Increasing the microbial CO2-fixing efficiency often requires supplying sufficient ATP and redirecting carbon flux for the production of metabolites. However, addressing these two issues concurrently remains a challenge. Here, we present a combinational strategy based on a synergetic CO2-fixing pathway that combines an ATP-generating carboxylation reaction in the central metabolic pathway with the ATP-consuming RuBisCO shunt in the carbon fixation pathway. This strategy provides enough ATP to improve the efficiency of CO2 fixation and simultaneously rewires the CO2-fixing pathway to the central metabolic pathway for the biosynthesis of chemicals. We demonstrate the application of this strategy by increasing the CO2-fixing rate and malate production in the autotroph Synechococcus elongatus by 110% and to 260 µM respectively, as well as increasing these two factors in the heterotrophic CO2-fixing Escherichia coli by 870% and to 387 mM respectively.

Enhancement of malate production through engineering of the periplasmic rTCA pathway in Escherichia coli.

The compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency; however, prokaryotes are unicellular organisms that lack membrane-bound organelles. To mimic this natural compartmentalization, we present here the targeting of the reductive tricarboxylic acid (rTCA) pathway to the periplasm to enhance the production of malate. A multigene combination knockout strategy was used to construct a phosphoenolpyruvate (PEP) pool. Then, the genes encoding phosphoenolpyruvate carboxykinase and malate dehydrogenase were combinatorially overexpressed to construct a cytoplasmic rTCA pathway for malate biosynthesis; however, the efficiency of malate production was low. To further enhance malate production, the rTCA pathway was targeted to the periplasm, which led to a 100% increase in malate production to 18.8 mM. Next, dual metabolic engineering regulation was adopted to balance the cytoplasmic and periplasmic pathways, leading to an increase in malate production to 58.8 mM. The final engineered strain, GL2306, produced 193 mM malate with a yield of 0.53 mol/mol in 5 L of pH-stat fed-batch culture. The strategy described here paves the way for the development of metabolic engineering and synthetic biology in the microbial production of chemicals.

Engineering Escherichia coli for malate production by integrating modular pathway characterization with CRISPRi-guided multiplexed metabolic tuning.

The application of rational design in reallocating metabolic flux to overproduce desired chemicals is always restricted by the native regulatory network. Here, we demonstrated that in vitro modular pathway optimization combined with in vivo multiplexed combinatorial engineering enables effective characterization of the bottleneck of a complex biosynthetic cascade and improves the output of the engineered pathway. As a proof of concept, we systematically identified the rate-limiting step of a five-gene malate biosynthetic pathway by combinatorially tuning the enzyme loads of a reconstituted biocatalytic reaction in a cell-free system. Using multiplexed CRISPR interference, we subsequently eliminated the metabolic constraints by rationally assigning an optimal gene expression pattern for each pathway module. The present engineered strain Escherichia coli B0013-47 exhibited a 2.3-fold increase in malate titer compared with that of the parental strain, with a yield of 0.85 mol/mol glucose in shake-flask culture and titer of 269 mM (36 g/L) in fed-batch cultivation. The strategy reported herein represents a powerful method for improving the efficiency of multi-gene pathways and advancing the success of metabolic engineering.

The resurrection genome of Boea hygrometrica: A blueprint for survival of dehydration.

"Drying without dying" is an essential trait in land plant evolution. Unraveling how a unique group of angiosperms, the Resurrection Plants, survive desiccation of their leaves and roots has been hampered by the lack of a foundational genome perspective. Here we report the ∼1,691-Mb sequenced genome of Boea hygrometrica, an important resurrection plant model. The sequence revealed evidence for two historical genome-wide duplication events, a compliment of 49,374 protein-coding genes, 29.15% of which are unique (orphan) to Boea and 20% of which (9,888) significantly respond to desiccation at the transcript level. Expansion of early light-inducible protein (ELIP) and 5S rRNA genes highlights the importance of the protection of the photosynthetic apparatus during drying and the rapid resumption of protein synthesis in the resurrection capability of Boea. Transcriptome analysis reveals extensive alternative splicing of transcripts and a focus on cellular protection strategies. The lack of desiccation tolerance-specific genome organizational features suggests the resurrection phenotype evolved mainly by an alteration in the control of dehydration response genes.

Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production.

L-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the L-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for L-malic acid biosynthesis. Second, the L-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the L-malic acid efflux system. Finally, the L-malic acid pathway was optimized by controlling gene expression levels, and the final L-malic acid concentration, yield, and productivity were up to 30.25 g L-1, 0.30 g g-1, and 0.32 g L-1 h-1 in the resulting strain W4209 with CaCO3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L-1, 0.31 g g-1, and 0.32 g L-1 h-1, respectively. The metabolic engineering strategy used here will be useful for efficient production of L-malic acid and other chemicals.

Systems metabolic engineering of Escherichia coli for high-yield production of Para-hydroxybenzoic acid.

Para-hydroxybenzoic acid (PHBA) is extensively used as an additive in the food and cosmetics industries, significantly enhancing product shelf life and stability. While microbial fermentation offers an environment-friendly and sustainable method for producing PHBA, the titer and productivity are limited due to product toxicity and complex metabolic flux distributions. Here, we initially redesigned a L-phenylalanine-producing Escherichia coli by employing rational metabolic engineering strategies, resulting in the production of PHBA reached the highest reported level of 14.17 g/L. Subsequently, a novel accelerated evolution system was devised comprising deaminase, the alpha subunit of RNA polymerase, an uracil-DNA glycosylase inhibitor, and the PHBA-responsive promoter PyhcN. This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC50) for PHBA within 15 days. Finally, the evolved strain achieved a production of 21.35 g/L PHBA in a 5-L fermenter, with a yield of 0.19 g/g glucose and a productivity rate of 0.44 g/L/h. This engineered strain emerges as a promising candidate for industrial production of PHBA through an eco-friendly approach.

Mechanism-Guided Computational Design Drives meso-Diaminopimelate Dehydrogenase to Efficient Synthesis of Aromatic d-amino Acids.

Aromatic d-amino acids (d-AAs) play a pivotal role as important chiral building blocks and key intermediates in fine chemical and drug synthesis. Meso-diaminopimelate dehydrogenase (DAPDH) serves as an excellent biocatalyst in the synthesis of d-AAs and their derivatives. However, its strict substrate specificity and the lack of efficient engineering methods have hindered its widespread application. Therefore, this study aims to elucidate the catalytic mechanism underlying DAPDH from Proteus vulgaris (PvDAPDH) through the examination of its crystallographic structure, computational simulations of potential energies and molecular dynamics simulations, and site-directed mutagenesis. Mechanism-guided computational design showed that the optimal mutant PvDAPDH-M3 increased specific activity and catalytic efficiency (kcat/Km) for aromatic keto acids up to 124-fold and 92.4-fold, respectively, compared to that of the wild type. Additionally, it expanded the substrate scope to 10 aromatic keto acid substrates. Finally, six high-value-added aromatic d-AAs and their derivatives were synthesized using a one-pot three-enzyme cascade reaction, exhibiting a good conversion rate ranging from 32 to 84% and excellent stereoselectivity (enantiomeric excess >99%). These findings provide a potential synthetic pathway for the green industrial production of aromatic d-AAs.

Rational Design of Salmonella typhi Acid Phosphatase for Efficient Production of Pyridoxal 5'-Phosphate.

Pyridoxal 5'-phosphate (PLP) is highly valuable in food and medicine. However, achieving the efficient biosynthesis of PLP remains challenging. Here, a salvage pathway using acid phosphatase from Salmonella typhi (StAPase) and pyridoxine oxidase from Escherichia coli (EcPNPO) as pathway enzymes was established for the first time to synthesize PLP from pyridoxine (PN) and pyrophosphate (PPi). StAPase was identified as a rate-limiting enzyme. Two protein modification strategies were developed based on the PN phosphorylation mechanism: (1) improving the binding of PN into StAPase and (2) enhancing the hydrophobicity of StAPase's substrate binding pocket. The kcat/Km of optimal mutant M7 was 4.9 times higher than that of the wild type. The detailed mechanism of performance improvement was analyzed. Under the catalysis of M7 and EcPNPO, a PLP high-yielding strain of 14.5 ± 0.55 g/L was engineered with a productivity of 1.0 ± 0.02 g/(L h) (the highest to date). The study suggests a promising method for industrial-scale PLP production.