Abstract:The background of developing cell factories for biorefinery was reviewed. Seventeen papers published in this special issue, covering the molecular mechanism of sugar utilization; genome-scale metabolic and regulative networks, the construction technologies, and the optimization of cell factories for biorefinery, were introduced.

Abstract:Lignocellulose is the most abundant natural biomass. Bioconversion of lignocelluloses becomes a bottleneck for biorefinery, because of its complex structures and heterogeneous composition. Besides screening or engineering approach for single free enzymes with improved properties, an alternative approach is to study synergistic pattern with hydrolysis systems or mimic natural cellulosome for better performance in cellulolytic substrate degradation. Besides, bacterial co-cultures provide another synergistic cellulolytic system. Engineered strains with modified metabolic network could facilitate consolidated bioprocess by increasing yields as well as reducing costs.

Abstract:Inefficient degradation of lignocellulose is one of the main barriers for the utilization of renewable plant biomass for biofuel production. The bottleneck of the biorefinery process is the generation of fermentable sugars from complicated biomass polymers. In nature, the main microbes of lignocelluloses deconstruction are fungi. Therefore, elucidating the mechanism of lignocelluloses degradation by fungi is of critical importance for the commercialization of lignocellulosic biofuels. This review focuses on the progress in lignocelluloses degradation pathways in fungi, especially on the advances made by functional genomics studies.

Abstract:Dozens of genome-scale metabolic networks have been reconstructed by integrating information from various databases on genes, proteins, metabolites and validated by experiment data from the literature. The reconstructed networks can be used to quantitatively investigate the interactions between components of a biological system at a system level. Such theoretical study could help us understand the organization principle of the large scale network and thus provide guidance to strain optimization through metabolic engineering technology. In this review, we evaluate the methods for the reconstruction, analysis and application of genome-scale metabolic networks. The difficulties and perspectives on this emerging research field are also discussed.

Abstract:A large and growing number of complete genomes from diverse species open tremendous opportunities for getting deep insights into cell metabolism. This increased understanding strongly supports engineering of cell metabolism for microbial production. In spite of the recent progress, a large fraction of genes in most of the available genomes remain incorrectly or imprecisely annotated. In this paper we review some of the new comparative genomics techniques used to reconstruct regulatory and metabolic networks from genomic data, reveal gaps in current knowledge, and identify previously uncharacterized genes. The application will be discussed by using a recent example–reconstruction of xylose utilization pathway in Clostridium acetobutylicum.

Abstract:Protein phosphorylation in bacteria is important for signaling and metabolic activity. Clostridium acetobutyicum can synthesize high yield of organic solvent under acidic condition. How solventogenesis is regulated at molecular level in this bacterium, is not clearly elucidated yet. We used two dimensional electrophoresis (2-DE) and mass spectrometry to have a differential analysis of the bacterial proteins from Clostridium acetobutylicum at acedogenic and solventogenic stage. We focused on these iso-spots with similar molecular mass and different pI values. Totally, eight string spots were identified, which displayed significant changes of pI values as well as spot volumes in response to solventogenic development. The data acquired from mass spectrometry demonstrated that all of the iso-spots contained the phosphrylated peptides. Bioinformatic analysis revealed that these proteins partake in the pathways of solvent synthesis.

Abstract:Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA ) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity.

Abstract:Clostridium acetobutylicum, a biofuel-butanol producer, has attracted worldwide interests. Strain improvement is important for the process of biobutanol industrialization where efficient genetic modification systems are essential. In this review, the history of genetic modification systems of C. acetobutylicum was introduced, and the types and principles of these systems and their disadvantages are summarized and analysed. The development of updated genetic modification systems for C. acetobutylicum is also proposed.

Abstract:Sepiolite?an inexpensive, resourceful, fibrous yet inoffensive mineral?made DNA transformation rapid, simple and efficient but the mechanism for DNA transformation was still unclear. Through RNA competition test, we proposed the different transforming mechanisms from the previous report. Meanwhile, we optimized the transforming method and could transfer a colony stored at 4°C for a month with plasmid through sepiolite fibers. The cells could be transformed well without competent cells preparation or incubation process. In sum, this was a novel potential transforming method, which could be explored further if the chemical method and electroporation could not be used.

Abstract:Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53×10?4, 1.64×10?4, 4.47×10?3, respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10?3 and 10?2.

Abstract:Bacterial promoter is a kind of regulators which are needed in bacterial gene expression and decide the strength and opportunity of gene expression. By insertion or deletion of promoters, we can change bacterial gene expression in order to study the growth and metabolic regulation. Promoters are also used to construct many kinds of vectors, so as to express heterologous genes. The study of promoter recognition and application is of great importance to realize the regulation of genes, gain products effectively and promote biological catalysis and metabolic engineering. This paper reviews bacterial promoters, and the methods for recognition of bacterial promoters as well as the study and application of bacterial promoters.

Abstract:Spore surface display is one of attractive microorganism surface display systems. With the advantage of resistance attribute and specific assembly pattern, the technology of spore surface display now is attracting more and more attention. According to the current reports and main achievements of spore surface display, the structure and assembly of spores, the principle for construction and some existing spore surface display systems were elaborated in this paper. Now with the unique property of spores, the technique is not only widely used in production of vaccines but also has great applied potential in the field of biocatalysis and cell-factory.

Abstract:Aspergillus niger is an important industrial workhorse with extensive application in the sectors of industrial enzymes, heterogeneous proteins, organic acids and etc. The disclosure of its genomic sequence to the public brought the study of A. niger into the post-genomic era. Diverse omic data are being produced massively and rapidly, which largely upgrades our understanding to the hyperproduction mechanism of A. niger to a systems and molecular level. At meanwhile, its genetic operating system is becoming mature, which enables genome-scale genetic perturbation within A. niger. In conclusion, we are on the right way to redesign and engineer A. niger to an omnipotent cellular factory.

Abstract:Microbial metabolic engineering and synthetic biology are important disciplines of microbial technology nowadays. Microbial cells are fast growing, easy to be cultivated in large scale, clear in genetic background and convenient in genetic modification. They play an important role in many domains. Microbial cell factory means an artificial microbial metabolic system that can be used in chemical production. The construction of a microbial cell factory needs transferring of multiple genes or a whole metabolic pathway, which may cause some problems such as metabolism imbalance and accumulation of mesostates. This review focuses on the regulation strategies of different levels involving simultaneous engagement of multiple genes. Future perspectives on the development of this domain were also discussed.

Abstract:Polyhydroxyalkanoates (PHA) are diverse polyesters synthesized by a variety of bacteria as intracellular carbon and energy storage compounds. As bio-renewable and biodegradable materials with diverse properties, PHA have drawn industrial attentions for their potential applications in many fields. This review focuses on recent strain developments for PHA production via cofactor engineering and metabolic engineering. The microaerobic production of PHA and application of PHA synthesis genes for improving robustness of industrial microorganisms are addressed.

Abstract:Discovery of an efficient bioconversion of cellulosic biomass and its hydrolysis to ethanol is the key to unlocking in developing of a bioethanol industry. The lack of industrially suitable microorganisms to convert xylose to ethanol fuel has been cited as a major technical bottleneck. In the past decades, many improvements have been made in the metabolic engineering of microorganisms, including Zymomonas mobilis, Escherichia coli, and yeasts, for the fermentation of xylose to produce ethanol by introducing genes for either xylose metabolism or ethanol production. The history and the current progress in constructing these strains are presented in this review.

Abstract:Sodium cellulose sulfate (NaCS)/Ploy-dimethyl-dially-ammonium-chloride (PDMDAAC) microcapsules were used as a novel pseudo “Cell Factory” to immobilize mixed bacteria for hydrogen production under anaerobic conditions. Compared to free cells, the hydrogen production was increased more than 30% with NaCS/PDMDAAC microcapsules as the pseudo “Cell Factory”. The biomass was increased from 1.5 g/L in free cell culture to 3.2 g/L in the pseudo “Cell Factory”. This pseudo “Cell Factory” system showed the excellent stability during 15 repeated-batches. The hydrogen yield maintained 1.73?1.81 mol H2/mol glucose. The fermentation cycle was shortened from 48 h to 24 h, resulting in an increase of 198.6% in the hydrogen production rate. There were high percentage of butyric acid and acetic acid in the culture broth, which meant that the pseudo “Cell Factory” established in the present work could be used for the multi-product system.

Abstract:To examine the substrate specificity of carotenoid 3',4'-desaturase (DR2250) from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBIEu and (or) pRK-CRTC and (or) pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 (1').