Abstract:The background and the importance of developing industrial biotechnology were illustrated, followed by a brief analysis on the driving effect of genomics and functional genomics. Seventeen papers covering metabolic engineering, fermentation engineering, industrial enzymes and biocatalysis are published in this special issue. These papers were briefly introduced to show the most recent developments of industrial biotechnology.

Abstract:The exploitation of microbial manufacture process (MMP) in industrial biotechnology requires a comprehensive understanding and an efficient modification of microorganism physiology. The availability of genome sequences and accumulation of -omics data allow us to understand of microbial physiology at the systems level, and genome-scale metabolic model (GSMM) represents a valuable framework for integrative analysis of metabolism of microorganisms. Genome scale metabolic models are reconstructed based on a combination of genome sequence and the more detailed biochemical knowledge, and these reconstructed models can be used for analyzing and simulating the operation of metabolism in response to different perturbations. Here we describe the reconstruction protocols for GSMM in further detail and provide the perspective of GSMM.

Abstract:Omics technologies have profoundly promoted development and applications of metabolic engineering by analysis of cell metabolism at a system level. Whole genome transcription profiles have provided researchers more rigorous evaluation of cell phenotype and an increased understanding of cellular metabolism. Furthermore, transcriptome analysis can conduce to identification of effective gene targets for strain improvement, and consequently accelerates rational design and construction of microbial cell factories for desired product. In this review, we briefly introduced the principle of three main platforms of transcriptome, and reviewed the recent applications of the transcriptome to metabolic engineering, finally provided conclusions and future prospects.

Abstract:Gene knockout, an important technology in molecular biology, has been broadly applied in industrial microbial metabolic engineering. From the basic mechanism of DNA recombination, we summarized and compared in this review different gene knockout strategies. Three most hot and important approaches, including the λ Red recombination system using the linear dsDNA as recombination substrate, the single or double crossover homologous recombination using the circular plasmid DNA as substrate, and the transposase mediated transposition recombination, were summarized in detail. Developing frontiers and application prospects of gene knockout were further discussed.

Abstract:Metabolic engineering is a technologic platform for industrial strain improvement and aims not only at modifying microbial metabolic fluxes, but also improving the physiological performance of industrial microbes. Microbes will meet multiple stresses in industrial processes. Consequently, elicited gene responses might result in a decrease in overall cell fitness and the efficiency of biotransformation. Thus, it is crucial to develop robust and productive microbial strains that can be integrated into industrial-scale bioprocesses. In this review, we focus on the progress of these novel methods and strategies for engineering stress-tolerance phenotypes referring to rational metabolic engineering and inverse metabolic engineering in recent years. In addition, we also address problems existing in this area and future research needs of microbial physiological functionality engineering.

Abstract:1,3-Dihydroxyacetone is widely used in cosmetics, medicines and food products. We reviewed the recent progress in metabolic pathways, key enzymes, as well as metabolic engineering for microbial production of 1,3-dihydroxyacetone. We addressed the research trend to increase yield of 1,3-dihydroxyacetone by improving the activity of glycerol dehydrogenase with genetic engineering, and regulating of fermentation process based on metabolic characteristic of the strain.

Abstract:Docosahexenoic acid (DHA) is an important polyunsaturated fatty acid which is beneficial to human health. Compared with the DHA derived from fish oil, DHA by microbial production possesses many advantages, and has a bright prospect. In this article, we reviewed strains, metabolic pathway, key enzymes and mechanism of lipid accumulation for microbial production of DHA. Those information would be greatly helpful for further improving DHA production by metabolic engineering.

Abstract:Arachidonic acid, as an important polyunsaturated fatty acid, is identified as potential food additives or pharmaceuticals for their biological activities. In recent years, arachidonic acid production by Mortierella alpina is becoming a research highlight. The prophase relevant researches focused on the mutagenic breeding and fermentation optimization. With the depth of investigation, the advancement concerning pathway for the biosynthesis of arachidonic acid in Mortierella alpina has been made. In this review, we summarized the prophase work briefly. Mainly, we discussed the biosynthesis pathway of arachidonic acid, the key enzymes, the construction of transformation system and the genetic modification. In addition, the prospect of microorganism arachidonic acid production is put forward.

Abstract:To investigate the energy utilization efficiency of Synechococcus sp. PCC7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and acetic acid respectively and discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could better enhance the growth of Synechococcus sp. PCC7942, and the latter was more effective. The metabolic flux through glycolytic pathway in mixotrophic cultures was stimulated by glucose whereas depressed by acetate, while the flux through the tricarboxylic acid cycle increased in both cases. Under mixotrophic conditions, glucose makes more significant impact on the diminishment of photochemical efficiency of Synechococcus sp. PCC7942. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher comparing with photoautotrophic culture. The energy conversion efficiencies based on ATP synthesis in photoautotrophic culture, mixotrophic cultures with glucose and with acetate were evaluated to be 6.81%, 7.43% and 8.77%, respectively.

Abstract:We investigated the effect of adding intermediate metabolites on cell growth and succinate production. The yield of succinic acid achieved to the highest when 0.5 g/L phosphoenolpyruvic acid (PEP) was added. According to the metabolic network of Actinobaccilus succinogenes NJ113, the metabolic flux was calculated by metabolic flux analysis. The ratio of hexose monophosphate pathway to glycolytic pathway increased from 39.4:60.3 to 76.8:22.6 after adding 0.5 g/L PEP, thus the reducing power was better balanced. The flux of PEP to oxaloacetate was 23.8% higher, which made the succinic acid flux improve from 99.8 mmol/(g DCW·h) to 124.4 mmol/(g DCW·h) and the flux of acetic acid and formic acid decreased by 22.9% and 15.4%, respectively. The key enzyme activity analysis showed that the specific activity of PEP carboxykinase reached to 1910 U/mg with 0.5 g/L PEP addition, which was 74.7% higher than the control; and the specific activity of pyruvate kinase decreased by 67.5%. Finally, the concentration of succinic acid was 29.1 g/L with the yield of 76.2%.

Abstract:Based on the fermentation analysis of Escherichia coli strains and cheap renewable resources suitable for polyhydroxybutyrate (PHB) production, we constructed a ptsG mutant of Escherichia coli DH5α. Application of E. coli DH5α mutant together with stress-induced system, we could produce PHB efficiently from cheap renewable sugar mixture by the simultaneous consumption of different sugars. Batch fermentation at lab scale (5 liter) showed that E. coli DH5α ΔptsG/pQKZ103 produced PHB from sugar mixture up to 84.6% of cell dry weight in 32 hours; meanwhile, the cell dry weight reached 8.24 g/L.

Abstract:The aim of this study was to improve the 2-keto-L-gulonic acid (2-KLG) production efficiency by Ketogulonicigenium vulgare and Bacillus megaterium by using multi-stage pH control strategy. The effect of pH on the cell growths and 2-KLG production showed that the optimum pH for K. vulgare and B. megaterium cell growth were 6.0 and 8.0, respectively, while the optimum pH for 2-KLG production was 7.0. Based on the above results, we developed a three-stage pH control strategy: the pH was kept at 8.0 during the first 8 h, then decreased to 6.0 for the following 12 h, and maintained at 7.0 to the end of fermentation. With this strategy, the titer, productivity of 2-KLG and L-sorbose consumption rate were achieved at 77.3 g/L, 1.38 g/(L·h) and 1.42 g/(L·h), respectively, which were 9.7%, 33.2% and 25.7% higher than the corresponding values of the single pH (pH 7.0) control model.

Abstract:We optimized the conditions of mixed fermentation of very high gravity ethanol with cassava flour and sugarcane juice. Based on the single factor experiment, we screened the important parameters for very high gravity ethanol fermentation with cassava flour and sugarcane juice by the Plackeet-burman design. Then, we obtained the optimum values of the important parameters by the orthogonal experiments: the mixing ratio of cassava flour to sugarcane juice, 1:5; initial pH of fermentation, 4.0?4.5; the concentrations of urea and MgSO4, 0.25% and 0.04% (W/W), respectively. Finally, we used a gradient temperature control strategy with the optimized conditions, and ethanol concentration of 17.84% (V/V) and fermentation efficiency of 91.82% were achieved, correspondingly.

Abstract:Spent cells recovered from anaerobic fermentation by Actinobacillus succinogenes were used as nitrogen source for succinic acid production. Three methods were investigated for cell wall-breaking. The results showed that enzymatic hydrolysis was more effective for higher succinic acid yield. When the enzymatic hydrolysate of spent cells was added to reach a total nitrogen concentration 1.11 g/L (equivalent to 10 g/L yeast extract), the succinic acid concentration was 42.0 g/L, but it increased slightly when enhancing the level of enzymatic hydrolysate. However, when 5 g/L yeast extract was supplemented with the enzymatic hydrolysate of spent cells, the succinic acid concentration reached 75.5 g/L after 36 hours and, the succinic acid productivity was 2.10 g/(L·h), which increased by 66.7% compared with the fermentation using 10 g/L yeast extract. Therefore, enzymatic hydrolysate of spent cells could replace 50% yeast extract in the original medium for succinic acid production.

Abstract:Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chy1 with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.

Abstract:The thrombolytic agent DSPAα1 is currently undergoing clinical trials for the treatment of acute ischemic stroke and has shown good pharmacodynamic, pharmacokinetic and safety profiles. Here, the DSPAα1 gene, optimized for the preferred codons of yeast, was cloned into the Pichia pastoris strains GS115 and KM71. Both expression systems produced functional DSPAα1 into the broth medium under shaking flask growth conditions with the yield of about 70 mg/L, and 105 mg/L, respectively. In addition, three glycosylation minus DSPAα1 mutants, constructed by site-directed mutagenesis, were also expressed in Pichia pastoris. The mutant proteins were assayed by SDS-PAGE and fibrin degradation activities were evaluated. The secretion levels of all the mutants, especially N362Q and N117Q/N362Q, were so lower compared to the wild-type DSPAα1 that only minimal quantities of mutant protein could be recovered by purification from the culture medium. The protein specific activities from mutants (N117Q, N362Q) were less 25% than that of the wild type protein. These results imply that the N-linked carbohydrate chains (at N117 and N362) are vital for the enzymatic activity of rDSPAα1 and for its secretion from Pichia pastoris.

Abstract:In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the β-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2°C, 5.5°C and 3.5°C, respectively, the halftime (t1/2, 60°C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the Vmax of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while Km of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the β-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of β-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.

Abstract:Hydroxymethyltransferase (SHMT) and tryptophanase (TPase) are key enzymes in biosynthesis of L-tryptophan. We constructed three recombinant plasmids, including pET-SHMT, pET-TPase, and pET-ST for over-expression or co-expression of SHMT and TPase in Escherichia coli BL21 (DE3). The SDS-PAGE analysis showed that the recombinant proteins of 47 kDa and 50 kDa were expressed of pET-SHMT and pET-TPase, respectively. As compared to the host stain, the enzyme activity of SHMT and TPase was increased by 6.4 and 8.4 folds, respectively. Co-expression of both recombinant proteins, 47 kDa and 50 kDa, was also successful by using pET-ST and the enzyme activities were enhanced by 6.1 and 6.9 folds. We designed two pathways of dual-enzymatic synthesis of L-tryptophan by using these recombinant strains as source of SHMT and TPase. In the first pathway, the pET-SHMT carrying strain was used to catalyze synthesis of L-serine, which was further transformed into L-tryptophan by the pET-TPase expressing strain. These two steps sequentially took place in different bioreactors. In the second pathway, the pET-ST carrying strain, in which two enzymes were co-expressed, was used to catalyze simultaneously two steps in a single bioreactor. HPLC analysis indicated a high yield of 41.5 g/L of L-tryptophan was achieved in the first pathway, while a lower yield of 28.9 g/L was observed in the second pathway. In the first pathway, the calculated conversion rates for L-glycine and indole were 83.3% and 92.5%, respectively. In the second pathway, a comparable conversion rate, 82.7%, was achieved for L-glycine, while conversion of indole was much lower, only 82.9%.