Jiangfeng Ma

Progress of succinic acid production from renewable resources: Metabolic and fermentative strategies

Succinic acid is a four-carbon dicarboxylic acid, which has attracted much interest due to its abroad usage as a precursor of many industrially important chemicals in the food, chemicals, and pharmaceutical industries. Facing the shortage of crude oil supply and demand of sustainable development, biological production of succinic acid from renewable resources has become a topic of worldwide interest. In recent decades, robust producing strain selection, metabolic engineering of model strains, and process optimization for succinic acid production have been developed. This review provides an overview of succinic acid producers and cultivation technology, highlight some of the successful metabolic engineering approaches.

Guidance for engineering of synthetic methylotrophy based on methanol metabolism in methylotrophy

Methanol is increasingly becoming an attractive substrate for production of different metabolites, such as commodity chemicals, and biofuels via biological conversion, due to the increment of annual production capacity and decrement of prices. In recent years, genetic engineering towards native menthol utilizing organisms – methylotrophy has developed rapidly and attracted widespread attention. Therefore, it is vital to elucidate the distinct pathways that involve methanol oxidation, formaldehyde assimilation and disassimilation in the different methylotrophies for future synthetic work. In addition, this will also help to genetically construct some new and non-native methylotrophies. This review summarizes the current knowledge about the methanol metabolism pathways in methylotrophy, discusses and compares different pathways on methanol utilization, and finally presents the strategies to integrate the methanol metabolism with other chemicals, biofuels or other high value-added product formation pathways.

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Abstract Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.

Enhanced biobutanol production with high yield from crude glycerol by acetone uncoupled Clostridium sp. strain CT7

This study reports a unique acetone uncoupled Clostridium species strain CT7, which shows efficient capability of glycerol utilization with high butanol ratio. Medium compositions, such as substrate concentration, micronutrients and pH show significant effects on butanol production from glycerol by strain CT7. To further maximize butanol production, fermentation conditions were optimized by using response surface methodology (RSM). Final butanol production of 16.6g/L with yield of 0.43g/g consumed glycerol was obtained, representing the highest butanol production and yield from glycerol in the batch fermentation mode. Furthermore, strain CT7 could directly convert crude glycerol to 11.8g/L of butanol without any pretreatment. Hence, strain CT7 shows immense potential for biofuels production using waste glycerol as cheap substrate.

Metabolic pathway involved in 6-chloro-2-benzoxazolinone degradation by Pigmentiphaga sp. strain DL-8 and identification of the novel metal-dependent hydrolase CbaA

ABSTRACT 6-Chloro-2-benzoxazolinone (CDHB) is a precursor of herbicide, insecticide, and fungicide synthesis and has a broad spectrum of biological activity. Pigmentiphaga sp. strain DL-8 can transform CDHB into 2-amino-5-chlorophenol (2A5CP), which it then utilizes as a carbon source for growth. The CDHB hydrolase (CbaA) was purified from strain DL-8, which can also hydrolyze 2-benzoxazolinone (BOA), 5-chloro-2-BOA, and benzamide. The specific activity of purified CbaA was 5,900 U · mg protein−1 for CDHB, with Km and k cat values of 0.29 mM and 8,500 s−1, respectively. The optimal pH for purified CbaA was 9.0, the highest activity was observed at 55°C, and the inactive metal-free enzyme could be reactivated by Mg2+, Ni2+, Ca2+, or Zn2+. Based on the results obtained for the CbaA peptide mass fingerprinting and draft genome sequence of strain DL-8, cbaA (encoding 339 amino acids) was cloned and expressed in Escherichia coli BL21(DE3). CbaA shared 18 to 21% identity with some metal-dependent hydrolases of the PF01499 family and contained the signature metal-binding motif Q127XXXQ131XD133XXXH137. The conserved amino acid residues His288 and Glu301 served as the proton donor and acceptor. E. coli BL21(DE3-pET-cbaA) resting cells could transform 0.2 mM CDHB into 2A5CP. The mutant strain DL-8ΔcbaA lost the ability to degrade CDHB but retained the ability to degrade 2A5CP, consistent with strain DL-8. These results indicated that cbaA was the key gene responsible for CDHB degradation by strain DL-8. IMPORTANCE 2-Benzoxazolinone (BOA) derivatives are widely used as synthetic intermediates and are also an important group of allelochemicals acting in response to tissue damage or pathogen attack in gramineous plants. However, the degradation mechanism of BOA derivatives by microorganisms is not clear. In the present study, we reported the identification of CbaA and metabolic pathway responsible for the degradation of CDHB in Pigmentiphaga sp. DL-8. This will provide microorganism and gene resources for the bioremediation of the environmental pollution caused by BOA derivatives.

Characterization of a β-glucosidase from Paenibacillus species and its application for succinic acid production from sugarcane bagasse hydrolysate

In this study, a β-glucosidase from Paenibacillus sp. M1 was expressed in E. coli BL21(DE3), purified and characterized. The specific activity of purified BglA was 137.64U·mg-1 protein with optimal temperature and pH of 50°C and 6.0. Furthermore, BglA shows excellent adaption to various environmental factors such as temperature, pH and metal ions. Engineered E. coli Suc260 was further reconstructed by overexpressing the β-glucosidase for achieving direct cellobiose utilization, which could efficiently utilize the pretreated sugarcane bagasses hydrolysate (SBH) consisting of 25.30g·L-1 cellobiose, 9.70g·L-1 glucose, 5.90g·L-1 arabinose and 7.10g·L-1 xylose. As a result, 26.50g·L-1 and 24.30g·L-1 succinic acid were produced by strain Suc260(pTbglA) from cellobiose and SBH with corresponding yields of 88.30% and 89.20% using dual-phase fermentation, respectively. This study indicated that incomplete enzymatic hydrolysate of SCB will be a potential feedstock for succinic acid production.

Current advance in biological production of malic acid using wild type and metabolic engineered strains

Malic acid (2-hydroxybutanedioic acid) is a four-carbon dicarboxylic acid, which has attracted great interest due to its wide usage as a precursor of many industrially important chemicals in the food, chemicals, and pharmaceutical industries. Several mature routes for malic acid production have been developed, such as chemical synthesis, enzymatic conversion and biological fermentation. With depletion of fossil fuels and concerns regarding environmental issues, biological production of malic acid has attracted more attention, which mainly consists of three pathways, namely non-oxidative pathway, oxidative pathway and glyoxylate cycle. In recent decades, metabolic engineering of model strains, and process optimization for malic acid production have been rapidly developed. Hence, this review comprehensively introduces an overview of malic acid producers and highlight some of the successful metabolic engineering approaches.

Expression and characterization of the key enzymes involved in 2-benzoxazolinone degradation by Pigmentiphaga sp. DL-8

In this study, the key enzymes involved in 2-benzoxazolinone (BOA) degradation by Pigmentiphaga sp. DL-8 were further verified and characterized in Escherichia coli. By codon optimization and co-expression of molecular chaperones in a combined strategy, recombinant BOA amidohydrolase (rCbaA) and 2-aminophenol (2-AP) 1,2-dioxygenase (rCnbCαCβ) were expressed and purified with the highest activity of 1934.6U·mgprotein-1 and 32.80U·mgprotein-1, respectively. BOA could be hydrolyzed to 2AP by rCbaA, which was further transformed to picolinic acid by rCnbCαCβ based on identified catalytic product. The optimal pH and temperature for rCbaA are 9.0 and 55°C with excellent stability for catalytic environments, and the residual activity was >50% after incubation at temperatures <45°C or at pH between 6.0 and 10.0 for 24h. On the contrary, rCnbCαCβ composed of α-subunit (33kDa) and β-subunit (38kDa) showed poor stability against environmental factors, including temperature, pH, metal ions and chemicals.

Ultrasound-assisted D-tartaric acid whole-cell bioconversion by recombinant Escherichia coli

d-Tartaric acid has wide range of application in the pharmaceutical industry and scarcely exists in nature. In this study, cis-epoxysuccinate hydrolase (CESH)-containing Escherichia coli was used to perform whole-cell bioconversion of cis-epoxysuccinate (CES) to D-tartaric acid and the catalytic efficiency was investigated by ultrasound treatment. The bioconversion rate of CES sodium reached 70.36% after 60 min treated after ultrasound, which is 3-fold higher than that in the control. The specific rate could be further improved by 2-fold after 5 repeated batches compared with the first one, however, the specific rate gradually decreased with the increase of repeat batches (>5 batches). The CESH from Bordetella sp. BK-52 was a typical Michaelis-Menten enzyme with Vmax and Km values of 28.17 mM/h/g WCW (wet of cell weight) and 30.18 mM, respectively. The process for the d-tartaric acid bioconversion, which consisted of 102.31 g/L CES sodium, 8.78 mg/mL whole cell and ultrasound power of 79.36 W, is further optimized using response surface methodology. The specific rate finally reached 194.79 ± 1.78 mM/h/g WCW under the optimal conditions. Furthermore, the permeability of inner and outer membrane was improved approximately 1.6 and 1.4-fold after ultrasound treatment, respectively, which may be a crucial factor for improvement of the bioconversion efficiency.

Biobutanol Production from Crystalline Cellulose through Consolidated Bioprocessing

Biobutanol production directly from lignocellulose, known as consolidated bioprocessing (CBP), is expected to be much less expensive than a process where hydrolytic enzyme production, cellulose saccharification, and microbial fermentation are accomplished separately. However, few microbes possess both cellulolytic and solventogenic properties in nature. Current research aims to endow cellulolytic microorganisms with butanol-producing ability or to set up microbial consortia for CBP. This review comprehensively details current achievements attempting to confer butanol-generating ability, not only to cellulolytic Clostridium strains but also to microbial consortia, to address and overcome major challenges in butanol production from cellulose. Recent advances in improving cellulosome activities within cellulolytic Clostridium strains are also emphasized.