Abstract:Biobased chemicals are one of the main missions of bioeconomy. In this special issue, we reviewed the recent progress in the metabolic engineering and fermentation control study on biobased succinic acid, adipic acid, lactic acid, 3-hydroxypropanoic acid, glucaric acid, glycerol, xylitol, higher alcohols and ethylene, recombinant construction for the direct utilization of lignocelluloses, biotransformation of bio-based lactic acid, and salting-out extraction of bio-based chemicals. Some research articles on biobased succinic acid, D-mannitol, malic acid, 5-aminolevulinic acid, 1,3-propanediol, and butanol are also included.

Abstract:Lignocellulosic biomass represents an abundant, low-cost and renewable source of potentially fermentable sugars. It is acandidate besides petroleum as feedstock for fuel and chemical production. Recent researches on utilizing lignocellulosicsas feedstock boost development of numerous-promising processes for a variety of fuels and chemicals, such as biodiesel, biohydrogen and ethanol. However, high cost in depolymerization is a primary obstacle preventing the use of lignocellulosic biomass as feedstock. Consolidated bioprocessing (CBP), refers to the bioprocess without any exogenous cellulolyotic enzymes added, converting the lignocellulosic material into biochemicals directly, which could potentially avoid the cost of the dedicated enzyme generation step by incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single organism through genetic engineering. There are two CBP strategies, native strategy and recombinant strategy. We mainly introduce the recombinant strategy, including its principle, the two responding styles, the contributions of synthetic biology and metabolic engineering and the future challenges.

Abstract:Confronted with the gradual exhaustion of the earth’s fossil energy resources and the grimmer environmental deterioration, the bio-based process to produce high-value added platform chemicals from renewable biomass is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to various advantages, such as 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. This review focuses on recent progresses in metabolic engineering of E. coli that lead to efficient recombinant biocatalysts for production of high-value organic acids such as succinic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like glycerol and xylitol. Besides, this review also discusses several other platform chemicals, including 2,5-furan dicarboxylic acid, aspartic acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxy-gamma-butyrolactone and sorbitol, which have not been produced by E. coli until now.

Abstract:Adipic acid is a six-carbon dicarboxylic acid, mainly for the production of polymers such as nylon, chemical fiber and engineering plastics. Its annual demand is close to 3 million tons worldwide. Currently, the industrial production of adipic acid is based on the oxidation of aromatics from non-renewable petroleum resources by chemo-catalytic processes. It is heavily polluted and unsustainable, and the possible alternative method for adipic acid production should be developed. In the past years, with the development of synthetic biology and metabolic engineering, green and clean biotechnological methods for adipic acid production attracted more attention. In this study, the research advances of adipic acid and its precursor production are reviewed, followed by addressing the perspective of the possible new pathways for adipic acid production.

Abstract:Succinic acid is one of the key intermediates in the tricarboxylic acid cycle (TCA)and has huge potentials in biopolymer, food, medicine applications. This article reviews recent research progress in the production of succinic acid by microbial fermentation, including discovery and screening of the succinic-acid-producing microbes, the progress of genetic engineering strategy and metabolic engineering technology for construction of succinic acid-producing strains, and fermentation process control and optimization. Finally, we discussed the limitation of current progress and proposed the future research needs for microbial production of succinic acid.

Abstract:Lactic acid is an important platform chemical. Especially with rapid development of poly (lactic acid) industry, the demand for L-lactic acid is continuously increasing. To further reduce the fermentation costs and improve the robustness of strains from industrial point of view, many modern biotechnological approaches have been applied to strain development. In this review, we briefly summarize recent advances in L-lactate fermentation by genetically modified microorganisms, including lactic acid bacteria, yeast, E. coli and Rhizopus species.

Abstract:Fermentative production of lactic acid, an important bio-based chemicals, has made considerable progress. In addition to the food industry and production of polylactic acid, lactic acid also can be used as an important platform chemical for the production of acrylic acid, pyruvic acid, 1,2-propanediol, and lactic acid esters. This article summarizes the recent progress in biocatalytic production of lactic acid derivatives by dehydration, dehydrogenation, reduction, and esterification. Trends in the biotransformation of lactic acid are also discussed.

Abstract:Higher alcohols have a high energy density, low hygroscopicity and can be mixed with gasoline at any ratio. It is the trend to?replace?fossil fuels with biofuels produced via microbial fermentation of renewable resources. We reviewed the progress in the development of engineered Saccharomyces cerevisiae and Escherichia coli that can produce higher alcohols, as well as the related technology platforms. We mainly focused on the construction of CoA-dependent pathways and α-keto acid mediated non-fermentative pathways, analyzed their respective characteristics, and summarized the construction strategies. The problems to be solved and future research direction were also discussed.

Abstract:Ethylene is the most widely used petrochemical feedstock globally. The development of bio-ethylene is essential due to limited fossil fuels and rising oil prices. Bio-ethylene is produced primarily by the dehydration of ethanol, but can alternatively be directly produced from ethylene biosynthesis pathways in plants, algae, or microorganisms by using cheap and renewable substrates. This review addressed the biosynthesis of ethylene in plants and microorganisms, the characterization of key enzymes, genetic engineering strategies for ethylene biosynthesis in microorganisms, and evaluated its perspective and successful cases toward the industrial application. The direct production of bio-ethylene from a biological process in situ is promising to supplement and even replace the petrochemical ethylene production.

Abstract:Bio-refinery using cheap biomass focuses mainly on strain improvement and fermentation strategies whereas less effort is made on down-stream processing. Using cheap biomass more impurities are introduced into the fermentation broths than mono-sugar substrate, thus down-stream processing for bio-based chemicals becomes the key problem in industrial production. The technique called salting-out extraction (SOE) was introduced in this review, which is used to separate target products from fermentation broth on the basis of partition difference of chemicals in two phases formed by mixing salts and organic solvents (or amphipathic chemicals) with broth at suitable ratios. The effect of solvents and salts on the formation of two aqueous phases, especially short chain alcohols and inorganic salts, and the application of SOE in recovery of bio-based chemicals, such as lactic acid, 1,3-propanediol, 2,3-butanediol and acetoin were summarized. The bio-chemicals were efficiently recovered from fermentation broth, and most of the impurities (cells and proteins) were removed in the same step. This technique is promising in the separation of bio-based chemicals, especially the recovery of hydrophilic molecules with low molecular weights.

Abstract:D-Mannitol has wide applications in food, pharmaceutical, and chemical industries. In this study, we constructed a genetically stable Escherichia coli strain for D-mannitol production by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC 12291 into chromosome of E. coli ATCC 8739 and inactivating other fermentation pathways (including pyruvate formate-lyase, lactate dehydrogenase, fumarate reductase, alcohol dehydrogenase, methylglyoxal synthase and pyruvate oxidase). Using mineral salts medium with glucose and fructose as carbon sources, the engineered strain could produce 1.2 mmol/L D-mannitol after anaerobic fermentation for 6 days. Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for 80 generations, D-mannitol titer increased 2.6-fold and mannitol dehydrogenase activity increased 2.8-fold. Genetically stable strains constructed in this work could ferment sugars to produce D-mannitol without the addition of antibiotics, inducers and formate, which was favorable for industrial production.

Abstract:Succinic acid production by fermentation from biomass, especially the lignocellulosic hydrolysate, is an alternative to chemical synthesis. Many studies report the inhibition of cell growth and succinic acid production from lignocellulosic hydrolysate, hardly is known about the actual kinetic and mechanism of the inhibition of individual factors. In this study, we studied inhibition effects of furfurals and 5-hydroxymethylfurfural (5-HMF) on cell growth and succinic acid production of engineered E. coli. Cell growth and succinic acid titer were severely inhibited by furfural and HMF with both concentrations higher than 0.8 g/L. Cell growth was totally inhibited when the concentration of furfural was above 6.4 g/L, or the concentration of HMF was above 12.8 g/L. At the concentration of maximum toleration, which was 3.2 g/L, furfural decreased the cell mass by 77.8% and the succinic acid titer by 36.1%. HMF decreased the cell mass by 13.6% and the succinic acid titer by 18.3%. Activity measurements of key enzymes revealed that phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarate reductase all were inhibited by furfural and HMF. This study gave a quantitative view to the succinic acid production under the inhibition of lignocellulose degradation products and will help overcome the difficulties of the lignocellulosic hydrolysate fermentation.

Abstract:Succinic acid is an important C4 platform chemical in the synthesis of many commodity and special chemicals. In the present work, different compounds were evaluated for succinic acid production by Actinobacillus succinogenes GXAS137. Important parameters were screened by the single factor experiment and Plackeet-Burman design. Subsequently, the highest production of succinic acid was approached by the path of steepest ascent. Then, the optimum values of the parameters were obtained by Box-Behnken design. The results show that the important parameters were glucose, yeast extract and MgCO3 concentrations. The optimum condition was as follows (g/L): glucose 70.00, yeast extract 9.20 and MgCO3 58.10. Succinic acid yield reached 47.64 g/L at the optimal condition. Succinic acid increased by 30% than that before the optimization (36.89 g/L). Response surface methodology was proven to be a powerful tool to optimize succinic acid production.

Abstract:Malic acid is widely used in food, and chemical industries. Through overexpressing pyruvate carboxylase and malate dehydrogenase in pdc1-deficient Saccharomyces cerevisiae, malic acid was successfully produced through the reductive TCA pathway. No malic acid was detected in wild type Saccharomyces cerevisiae, however, 45 mmol/L malic acid was produced in engineered strain, and the concentration of byproduct ethanol also reduced by 18%. The production of malic acid enhanced 6% by increasing the concentration of Ca2+. In addition, the final concentration reached 52.5 mmol /L malic acid by addition of biotin. The increasing is almost 16% higher than that of the original strain.

Abstract:5-aminolevulinic acid (ALA), a precursor for biosynthesis of pyrrole compounds in living organisms, has been widely used in agriculture and medical photodynamics therapy and is regarded as a promising value-added bio-based chemical. In the previous investigations on ALA production with recombinant Escherichia coli expressing heterogenous C4 pathway gene, LB media supplemented with glucose and ALA precursors succinate and glycine is widely used, leading to high production cost. Succinate participates in ALA biosynthesis in a form of succinyl-CoA. In this study, genes involved in succinyl-CoA consumption, sdhAB (encoding succinic dehydrogenase) or sucCD (encoding succinyl-CoA synthetase) of E. coli MG1655 was knocked out and tested for ALA accumulation. In comparison with the recombinant E. coli strain expressing heterogenous ALA synthetase, the sdhAB- or sucCD-deficient strain accumulate 25.59% and 12.40%, respectively, more ALA in a 5 L fermentor using a defined synthetic medium with glucose as main carbon source and without supplementation of succinate, providing a novel cost-effective approach for industrial production of ALA.

Abstract:Production of 1,3-propanediol, a byproduct of biodiesel production, is important to increase the economic benefit of biodiesel industry. Accumulation of 3-hydroxypropionaldehyde is one of the key problems in the 1,3-propanediol fermentation process, leading to the cell death and the fermentation abnormal ceasing. Different from the traditional way of reducing the accumulation of the 3-hydroxypropionaldehyde, we introduced the polyhydroxybutyrate pathway into the Klebsiella pneumoniae for the first time to enhance the tolerance of K. pneumoniae to 3-hydroxypropionaldehyde, at the same time, to improve the 1,3-propanediol production. Plasmid pDK containing phbC, phbA, phbB gene was constructed and transformed into K. pneumoniae successfully. PHB was detected in the engineered K. pneumoniae after IPTG induction and its content was enhanced with the IPTG concentration increasing. The optimized IPTG concentration was 0.5 mmol/L. The constructed K. pneumoniae could produce 1,3-propanediol normally, at the same time accumulate polyhydroxybutyrate. With the constructed strain, the fermentation proceeds normally; 1,3-propanediol concentration and yield reached 31.3 g/L and 43.9% at 72 h. Our work is helpful for the deep understanding of 1,3-propanediol metabolic mechanism of Klebsiella pneumoniae, and also provides a new way for strain optimization of Klebsiella pneumoniae.

Abstract:Butanol production from corn stover hydrolysates (CSH) with in-situ liquid-liquid extraction was studied to enhance the production and reduce the fermentation cost. Oleyl alcohol was selected as the suitable solvent and added at the initial fermentation time with the ratio of 1:1 (oleyl alcohol: fermentation broth, V/V). Under this condition, butanol and ABE from CSH with 32.1 g/L total sugars were 3.28 and 4.72 g/L, which were 958.1% and 742.9% higher than those of the controls, respectively. Butanol and ABE production from CSH of 49.7 g/L total sugars after detoxification by ion exchange resin D301 coupled with extraction fermentation were 10.34 g/L and 14.72 g/L with an ABE yield of 0.31 g/g (g ABE/g utilized sugar), which were equal to those of glucose and xylose mixture fermentation. The detoxification and extraction fermentation technology of cellulosic butanol production would provide a crucial technical support to the industrialized production of cellulosic butanol.