Yujin Cao

Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols

Confronted with the gradual and inescapable exhaustion of the earth’s fossil energy resources, the bio-based process to produce platform chemicals from renewable carbohydrates is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to its clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. Compared to previous reviews, this review focuses on recent advances in metabolic engineering of the industrial model bacteria E. coli that lead to efficient recombinant biocatalysts for the production of high-value organic acids like succinic acid, lactic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like 1,3-propanediol, xylitol, mannitol, and glycerol with the discussion of the future research in this area. Besides, this review also discusses several platform chemicals, including fumaric acid, aspartic acid, glutamic acid, sorbitol, itaconic acid, and 2,5-furan dicarboxylic acid, which have not been produced by E. coli until now.

Biosynthesis of phloroglucinol compounds in microorganisms—review

Phloroglucinol derivatives are a major class of secondary metabolites of wide occurrence in biological systems. In the bacteria kingdom, these compounds can only be synthesized by some species of Pseudomonads. Pseudomonas spp. could produce 2,4-diacetylphloroglucinol (DAPG) that plays an important role in the biological control of many plant pathogens. In this review, we summarize knowledge about synthesis of phloroglucinol compounds based on the DAPG biosynthetic pathway. Recent advances that have been made in understanding phloroglucinol compound biosynthesis and regulation are highlighted. From these studies, researchers have identified the biosynthesis pathway of DAPG. Most of the genes involved in the biosynthesis pathway have been cloned and characterized. Additionally, heterologous systems of the model microorganism Escherichia coli are constructed to produce phloroglucinol. Although further work is still required, a full understanding of phloroglucinol compound biosynthesis is almost within reach. This review also suggests new directions and attempts to gain some insights for better understanding of the biosynthesis and regulation of DAPG. The combination of traditional biochemistry and molecular biology with new systems biology and synthetic biology tools will provide a better view of phloroglucinol compound biosynthesis and a greater potential of microbial production.

Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase.

BackgroundThioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.ResultsWe cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.ConclusionsFor the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.

Fatty acid from the renewable sources: A promising feedstock for the production of biofuels and biobased chemicals

With the depletion of the nonrenewable petrochemical resources and the increasing concerns of environmental pollution globally, biofuels and biobased chemicals produced from the renewable resources appear to be of great strategic significance. The present review described the progress in the biosynthesis of fatty acid and its derivatives from renewable biomass and emphasized the importance of fatty acid serving as the platform chemical and feedstock for a variety of chemicals. Due to the low efficient conversions of lignocellulosic biomass or carbon dioxide to fatty acid, we also put forward that rational strategies for the production of fatty acid and its derivatives should further derive from the consideration of whole bioprocess (pretreatment, saccharification, fermentation, separation), multiscale analysis and interdisciplinary combinations (omics, kinetics, metabolic engineering, synthetic biology, fermentation and so on).

Fermentative succinate production: an emerging technology to replace the traditional petrochemical processes.

Succinate is a valuable platform chemical for multiple applications. Confronted with the exhaustion of fossil energy resources, fermentative succinate production from renewable biomass to replace the traditional petrochemical process is receiving an increasing amount of attention. During the past few years, the succinate-producing process using microbial fermentation has been made commercially available by the joint efforts of researchers in different fields. In this review, recent attempts and experiences devoted to reduce the production cost of biobased succinate are summarized, including strain improvement, fermentation engineering, and downstream processing. The key limitations and challenges faced in current microbial production systems are also proposed.

Metabolic engineering of Escherichia coli for the production of xylonate.

Xylonate is a valuable chemical for versatile applications. Although the chemical synthesis route and microbial conversion pathway were established decades ago, no commercial production of xylonate has been obtained so far. In this study, the industrially important microorganism Escherichia coli was engineered to produce xylonate from xylose. Through the coexpression of a xylose dehydrogenase (xdh) and a xylonolactonase (xylC) from Caulobacter crescentus, the recombinant strain could convert 1 g/L xylose to 0.84 g/L xylonate and 0.10 g/L xylonolactone after being induced for 12 h. Furthermore, the competitive pathway for xylose catabolism in E. coli was blocked by disrupting two genes (xylA and xylB) encoding xylose isomerase and xylulose kinase. Under fed-batch conditions, the finally engineered strain produced up to 27.3 g/L xylonate and 1.7 g/L xylonolactone from 30 g/L xylose, about 88% of the theoretical yield. These results suggest that the engineered E. coli strain has a promising perspective for large-scale production of xylonate.

Imidazolium-based ionic liquids for cellulose pretreatment: recent progresses and future perspectives

As the most abundant biomass in nature, cellulose is considered to be an excellent feedstock to produce renewable fuels and fine chemicals. Due to its hydrogen-bonded supramolecular structure, cellulose is hardly soluble in water and most conventional organic solvents, limiting its further applications. The emergence of ionic liquids (ILs) provides an environmentally friendly, biodegradable solvent system to dissolve cellulose. This review summarizes recent advances concerning imidazolium-based ILs for cellulose pretreatment. The structure of cations and anions which has an influence on the solubility is emphasized. Methods to assist cellulose pretreatment with ILs are discussed. The state of art of the recovery, regeneration, and reuse aspects of ILs is also presented in this work. The current challenges and development directions of cellulose dissolution in ILs are put forward. Although further studies are still much required, commercialization of IL-based processes has made great progress in recent years.

Biotechnological production of 1,2,4-butanetriol: An efficient process to synthesize energetic material precursor from renewable biomass

1,2,4-Butanetriol (BT) is a valuable chemical with extensive applications in many different fields. The traditional chemical routes to synthesize BT suffer from many drawbacks, e.g., harsh reaction conditions, multiple steps and poor selectivity, limiting its industrial production. In this study, an engineered Escherichia coli strain was constructed to produce BT from xylose, which is a major component of the lignocellulosic biomass. Through the coexpression of a xylose dehydrogenase (CCxylB) and a xylonolactonase (xylC) from Caulobacter crescentus, native E. coli xylonate dehydratase (yjhG), a 2-keto acid decarboxylase from Pseudomonas putida (mdlC) and native E. coli aldehyde reductase (adhP) in E. coli BL21 star(DE3), the recombinant strain could efficiently convert xylose to BT. Furthermore, the competitive pathway responsible for xylose metabolism in E. coli was blocked by disrupting two genes (xylA and EcxylB) encoding xylose isomerase and xyloluse kinase. Under fed-batch conditions, the engineered strain BL21ΔxylAB/pE-mdlCxylBC&pA-adhPyjhG produced up to 3.92 g/L of BT from 20 g/L of xylose, corresponding to a molar yield of 27.7%. These results suggest that the engineered E. coli has a promising prospect for the large-scale production of BT.

Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli

Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS-PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1Delta9) and cis-vaccenate (18:1Delta11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1Delta9) was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase.

Not only osmoprotectant: Betaine increased lactate dehydrogenase activity and L-lactate production in lactobacilli

Lactobacilli are commonly used for industrial production of polymer-grade L-lactic acid. The present study tested the Tween 80 alternative betaine in L-lactate production by several industrial lactobacilli. In flask fermentation of Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactis and Lactobacillus rhamnosus, the betaine addition (2g/l) had similar osmoprotectant effect with Tween 80 but had increased the lactate dehydrogenase activities and L-lactate production than Tween 80 control. In fed-batch fermentation of L. casei, betaine supplementation improved the L-lactic acid titer to 190 g/l, the yield to 95.5% (g L-lactic acid/g glucose), the productivity to 2.6g/lh, and the optical purity to 97.0%. The results demonstrated that supplementation of Tween 80 alternative - betaine in the fermentation medium is feasible for industrial l-lactic acid fermentation by lactobacilli, which will improve the lactate production but will not increase the process costs and modify any process conditions.