Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

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.

Rational Design of the Spatial Effect in a Fe(II)/α-Ketoglutarate-Dependent Dioxygenase Reverses the Regioselectivity of C(sp3)-H Bond Hydroxylation in Aliphatic Amino Acids.

The hydroxylation of remote C(sp3)-H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high-value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)-H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α-ketoglutarate-dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L-Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)-H bonds, but also provides a theoretical guidance for the precision-driven modification of similarly remote C(sp3)-H bonds in complex molecules.

Efficient Production of Epoxy-Norbornane from Norbornene by an Engineered P450 Peroxygenase.

Epoxy-norbornane (EPO-NBE) is a crucial building block for the synthesis of various biologically active heterocyclic systems. To develop an efficient protocol for producing EPO-NBE using norbornene (NBE) as a substrate, cytochrome P450 enzyme from Pseudomonas putida (CYP238A1) was examined and its crystal structure (PDB code: 7X53) was resolved. Molecular mechanism analysis showed a high energy barrier related to iron-alkoxy radical complex formation. Therefore, a protein engineering strategy was developed and an optimal CYP238A1NPV variant containing a local hydrophobic "fence" at the active site was obtained, which increased the H2 O2 -dependent epoxidation activity by 7.5-fold compared with that of CYP238A1WT . Among the "fence", Glu255 participates in an efficient proton transfer system. Whole-cell transformation using CYP238A1NPV achieved an EPO-NBE yield of 77.6 g ⋅ L-1 in a 30-L reactor with 66.3 % conversion. These results demonstrate the potential of this system for industrial production of EPO-NBE and provides a new biocatalytic platform for epoxidation chemistry.

A Tri-Enzyme Cascade for Efficient Production of L-2-Aminobutyrate from L-Threonine.

L-2-aminobutyrate (L-ABA) is an important chiral drug intermediate with a key role in modern medicinal chemistry. Here, we describe the development of an efficient method for the asymmetric synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using a cost-effective L-Thr. Low activity of leucine dehydrogenase from Bacillus thuringiensis (BtLDH) and unbalanced expression of enzymes in the cascade were major challenges. Mechanism-based protein engineering generated the optimal triple variant BtLDHM3 (A262S/V296C/P150M) with 20.7-fold increased specific activity and 9.6-fold increased kcat /Km compared with the wild type. Optimizing plasmids with different copy numbers regulated enzymatic expression, thereby increasing the activity ratio (0.3 : 1:0.6) of these enzymes in vivo close to the optimal ratio (0.4 : 1 : 1) in vitro. Importing the optimal triple mutant BtLDHM3 into our constructed pathway in vivo and optimization of transformation conditions achieved one-pot conversion of L-Thr to 130.2 g/L L-ABA, with 95 % conversion, 99 % e.e. and 10.9 g L-1 h-1 productivity (the highest to date) in 12 h on a 500 mL scale. These results describe a potential biosynthesis approach for the industrial production of L-ABA.

Systems engineering of Escherichia coli for high-level shikimate production.

To further increase the production efficiency of microbial shikimate, a valuable compound widely used in the pharmaceutical and chemical industries, ten key target genes contributing to shikimate production were identified by exploiting the enzyme constraint model ec_iML1515, and subsequently used for promoting metabolic flux towards shikimate biosynthesis in the tryptophan-overproducing strain Escherichia coli TRP0. The engineered E. coli SA05 produced 78.4 g/L shikimate via fed-batch fermentation. Deletion of quinate dehydrogenase and introduction of the hydroaromatic equilibration-alleviating shikimate dehydrogenase mutant AroET61W/L241I reduced the contents of byproducts quinate (7.5 g/L) and 3-dehydroshikimic acid (21.4 g/L) by 89.1% and 52.1%, respectively. Furthermore, a high concentration shikimate responsive promoter PrpoS was recruited to dynamically regulate the expression of the tolerance target ProV to enhance shikimate productivity by 23.2% (to 2 g/L/h). Finally, the shikimate titer was increased to 126.4 g/L, with a yield of 0.50 g/g glucose and productivity of 2.63 g/L/h, using a 30-L fermenter and the engineered strain E. coli SA09. This is, to the best of our knowledge, the highest reported shikimate titer and productivity in E. coli.

A Multi-Enzyme Cascade for Efficient Production of Pyrrolidone from l-Glutamate.

Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.

Rational Design of Meso-Diaminopimelate Dehydrogenase with Enhanced Reductive Amination Activity for Efficient Production of d-p-Hydroxyphenylglycine.

d-p-hydroxyphenylglycine (d-HPG) is an important intermediate in the pharmaceutical industry. In this study, a tri-enzyme cascade for the production of d-HPG from l-HPG was designed. However, the amination activity of Prevotella timonensis meso-diaminopimelate dehydrogenase (PtDAPDH) toward 4-hydroxyphenylglyoxylate (HPGA) was identified as the rate-limiting step. To overcome this issue, the crystal structure of PtDAPDH was solved, and a "binding pocket and conformation remodeling" strategy was developed to improve the catalytic activity toward HPGA. The best variant obtained, PtDAPDHM4, exhibited a catalytic efficiency (kcat/Km) that was 26.75-fold higher than that of the wild type. This improvement was due to the enlarged substrate-binding pocket and enhanced hydrogen bond networks around the active center; meanwhile, the increased number of interdomain residue interactions drove the conformation distribution toward the closed state. Under optimal transformation conditions, PtDAPDHM4 produced 19.8 g/L d-HPG from 40 g/L racemate DL-HPG in a 3 L fermenter within 10 h, with 49.5% conversion and >99% enantiomeric excess. Our study provides an efficient three-enzyme cascade pathway for the industrial production of d-HPG from racemate DL-HPG. IMPORTANCE d-p-hydroxyphenylglycine (d-HPG) is an important intermediate in the synthesis of antimicrobial compounds. d-HPG is mainly produced via chemical and enzymatic approaches, and enzymatic asymmetric amination employing diaminopimelate dehydrogenase (DAPDH) is considered an attractive method. However, the low catalytic activity of DAPDH toward bulky 2-keto acids limits its applications. In this study, we identified a DAPDH from Prevotella timonensis and created a mutant, PtDAPDHM4, which exhibited a catalytic efficiency (kcat/Km) toward 4-hydroxyphenylglyoxylate that was 26.75-fold higher than that of the wild type. The novel strategy developed in this study has practical value for the production of d-HPG from inexpensive racemate DL-HPG.

Protein engineering, cofactor engineering, and surface display engineering to achieve whole-cell catalytic production of chondroitin sulfate A.

Chondroitin sulfate A (CSA) is a valuable glycosaminoglycan that has great market demand. However, current synthetic methods are limited by requiring the expensive sulfate group donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and inefficient enzyme carbohydrate sulfotransferase 11 (CHST11). Herein, we report the design and integration of the PAPS synthesis and sulfotransferase pathways to realize whole-cell catalytic production of CSA. Using mechanism-based protein engineering, we improved the thermostability and catalytic efficiency of CHST11; its Tm and half-life increased by 6.9°C and 3.5 h, respectively, and its specific activity increased 2.1-fold. Via cofactor engineering, we designed a dual-cycle strategy of regenerating ATP and PAPS to increase the supply of PAPS. Through surface display engineering, we realized the outer membrane expression of CHST11 and constructed a whole-cell catalytic system of CSA production with an 89.5% conversion rate. This whole-cell catalytic process provides a promising method for the industrial production of CSA.

Improving tyrosol production efficiency through shortening the allosteric signal transmission distance of pyruvate decarboxylase.

Tyrosol is an important chemical in medicine and chemical industries, which can be synthesized by a four-enzyme cascade pathway constructed in our previous study. However, the low catalytic efficiency of pyruvate decarboxylase from Candida tropicalis (CtPDC) in this cascade is a rate-limiting step. In this study, we resolved the crystal structure of CtPDC and investigated the mechanism of allosteric substrate activation and decarboxylation of this enzyme toward 4-hydroxyphenylpyruvate (4-HPP). In addition, based on the molecular mechanism and structural dynamic changes, we conducted protein engineering of CtPDC to improve decarboxylation efficiency. The conversion of the best mutant, CtPDCQ112G/Q162H/G415S/I417V (CtPDCMu5), had over two-fold improvement compared to the wild-type. Molecular dynamic (MD) simulation revealed that the key catalytic distances and allosteric transmission pathways were shorter in CtPDCMu5 than in the wild type. Furthermore, when CtPDC in the tyrosol production cascade was replaced with CtPDCMu5, the tyrosol yield reached 38 g·L-1 with 99.6% conversion and 1.58 g·L-1·h-1 space-time yield in 24 h through further optimization of the conditions. Our study demonstrates that protein engineering of the rate-limiting enzyme in the tyrosol synthesis cascade provides an industrial-scale platform for the biocatalytic production of tyrosol. KEY POINTS: • Protein engineering of CtPDC based on allosteric regulation improved the catalytic efficiency of decarboxylation. • The application of the optimum mutant of CtPDC removed the rate-limiting bottleneck in the cascade. • The final titer of tyrosol reached 38 g·L-1 in 24 h in 3 L bioreactor.

Improving Theaflavin-3,3'-digallate Production Efficiency Optimization by Transition State Conformation of Polyphenol Oxidase.

Theaflavins (TFs) are good for health because of their bioactivities. Enzymatic synthesis of TFs has garnered much attention; however, the source and activity of the enzymes needed limit their wide application. In this study, a microbial polyphenol oxidase from Bacillus megaterium was screened for the synthesis of theaflavin-3,3'-digallate (TFDG). Based on structural and mechanistic analyses of the enzyme, the O-O bond dissociation was identified as the rate-determining step. To address this issue, a transition state (TS) conformation optimization strategy was adopted to stabilize the spatial conformation of the O-O bond dissociation, which improved the catalytic efficiency of tyrosinase. Under the optimum transformation conditions of pH 4.0, temperature 25 °C, (-)-epigallocatechin gallate/epicatechin gallate molar ratio of 2:1, and time of 30 min, Mu4 (BmTyrV218A/R209S) produced 960.36 mg/L TFDG with a 44.22% conversion rate, which was 6.35-fold higher than that of the wild type. Thus, the method established has great potential in the synthesis of TFDG and other TFs.

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production.

Microbial cell factories provide a green and sustainable opportunity to produce value-added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A-IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03-FM, the amounts of assimilated methanol and malate were increased by 162 % and 61 %, respectively. Furthermore, manipulating the MLO rigidity in strain ZP04-RB made acetyl-coA synthesis from oxidative glycolysis change to non-oxidative glycolysis; consequently, CO2 release decreased by 35 % and the n-butanol yield increased by 20 %. This artificial MLO provides a strategy for the co-localization of enzymes to channel C1 starting materials into value-added chemicals.

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.

Mediator Engineering of Saccharomyces cerevisiae To Improve Multidimensional Stress Tolerance.

Saccharomyces cerevisiae is a well-performing workhorse in chemical production, which encounters complex environmental stresses during industrial processes. We constructed a multiple stress tolerance mutant, Med15V76R/R84K, that was obtained by engineering the KIX domain of Mediator tail subunit Med15. Med15V76R/R84K interacted with transcription factor Hap5 to improve ARV1 expression for sterol homeostasis for decreasing membrane fluidity and thereby enhancing acid tolerance. Med15V76R/R84K interacted with transcription factor Mga2 to improve GIT1 expression for phospholipid biosynthesis for increasing membrane integrity and thereby improving oxidative tolerance. Med15V76R/R84K interacted with transcription factor Aft1 to improve NFT1 expression for inorganic ion transport for reducing membrane permeability and thereby enhancing osmotic tolerance. Based on this Med15 mutation, Med15V76R/R84K, the engineered S. cerevisiae strain, showed a 28.1% increase in pyruvate production in a 1.0-L bioreactor compared to that of S. cerevisiae with its native Med15. These results indicated that Mediator engineering provides a potential alternative for improving multidimensional stress tolerance in S. cerevisiae. IMPORTANCE This study identified the role of the KIX domain of Mediator tail subunit Med15 in response to acetic acid, H2O2, and NaCl in S. cerevisiae. Engineered KIX domain by protein engineering, the mutant strain Med15V76R/R84K, increased multidimensional stress tolerance and pyruvate production compared with that of S. cerevisiae with its native Med15. The Med15V76R/R84K could increase membrane related genes expression possibly by enhancing interaction with transcription factor to improve membrane physiological functions under stress conditions.

Engineering Escherichia coli asymmetry distribution-based synthetic consortium for shikimate production.

Microbial consortia constitute a promising tool for achieving high-value chemical bio-production. However, customizing the consortium ratio remains challenging. Herein, an asymmetry distribution-based synthetic consortium (ADSC) was developed to switch cell phenotypes using shikimate synthesis for proof of concept. First, the cell pole-organizing protein PopZ was screened as a mediator of asymmetric protein distribution in Escherichia coli. The ADSC was then constructed to incorporate PopZ-mediated asymmetry distribution and a TetR-based transcription repression switch to achieve the dynamical control of microbial population ratio. Finally, the ADSC was used to decouple cell growth from shikimate synthesis by effectively coordinating the ratio of growing cells and production cells at the consortium level, thereby increasing shikimate titer to 30.1 g/L in the 7.5-L bioreactor with a minimal medium. This titer was further improved to 82.5 g/L when using rich medium fermentation. Our results illustrate a novel approach to control consortium structure through ADSC-mediated regulation, highlighting its potential as an efficient strategy for controlling metabolic state in microbes.

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.

Advances in microbial synthesis of bioplastic monomers.

Bio-based plastics production offers an alternative to the environmental problems posed by a significant reliance on fossil fuels. While dicarboxylic acids were essential bioplastic monomers, producing them on a large scale proved problematic. Recently, metabolic engineering has opened up interesting possibilities for producing dicarboxylic acids sustainably and efficiently. In this chapter, studies on the development of several dicarboxylic acid bioplastic monomers were presented. Furthermore, for different dicarboxylic acids, a variety of metabolic engineering strategies were highlighted, including improving the utilization rate of substrates, strengthening the catalytic efficiency of key enzymes, blocking branching pathways to balance metabolic flux, and improving cell physiological performance to promote biosynthesis. Finally, the remaining obstacles and solutions for building advanced dicarboxylic acid microbial systems were discussed.