Biotechnology Journal

This regular issue of BTJ features articles on the production of biofuels, stem cells and gene expression. The cover is inspired by the article ”Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification“ by Robson Tramontina et al. which analyzed a mixture of several Aspergillus enzymes involved in the degradation of polysaccharide. © Dr_Kateryna – Fotolia.com

This review covers the current selection methods that are available to scientists in order to isolate a particular cell (”clone“) that produces the greatest amount of a protein of interest. Traditional and simple methods are reviewed, as well as more complex methods that utilize the latest advancements in automated equipment and technology. This review will serve as a guide and reference for researchers, and will allow them to determine which selection method best suits their particular needs.

Gene expression requires transcription factor proteins to bind to DNA promoter elements in order to initiate transcription. In this study, the authors present a coordinated approach that engineers both transcription factor and promoter elements to achieve strong and modular yeast expression systems. This resulted in a mutant transcription factor used to drive the expression of ultra-strong promoters and a modular expression system capable of staged outputs. This work expands the expression tools available in yeast and demonstrates the potential for engineering endogenous transcription factors and promoter elements.

New methodology was developed that allowed the packaging and screening of all microbial DNA in an environmental soil sample. In this paper, the DNA was screened for genes that conferred tolerance to 1-butanol, a potential biofuel that is highly toxic to fermenting cultures. Genes conferring greater tolerance were identified and Raman spectroscopy was used to determined how they changed the chemical composition of cells. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real-time and is adaptable to high-throughput screening, making it relevant to metabolic engineering.

A coupled enzyme reaction on biologically produced PhaC-particles for the production of chiral alcohols is provided. Alcohol dehydrogenase (ADH) generating the desired substance and formate dehydrogenase (FDH) regenerating the cofactor for the reaction are recombinantly produced in E. coli cells and loaded onto particles via coiled coil interaction. This strategy may become a cost-effective alternative to coupled reactions using purified enzymes.

Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This paper presents a systematic methodology which facilitates the selection of a technology option suitable for future primary recovery operation. The authors demonstrate the technology selection procedure by comparing current and alternative primary recovery technologies and combined the results with a multi-attribute decision making technique as well as economic analysis, identifying suitable technology candidates.

Cyanovirin-N (CV-N) is a lectin which has potential as an HIV microbicide candidate. CV-N produced by transgenic plants can be secreted out of the roots into hydroponic medium. The authors demonstrate that the yield of CV-N in the medium is increased by shaking the containers in which plants are cultured. They identify the proteins present in the hydroponic medium (the ‘rhizosecretome’) and demonstrate that a simple, inexpensive, ion exchange chromatography protocol can produce semi-purified CV-N from the medium, which is able to neutralise HIV.

Protein A chromatography is the main method used for capturing monoclonal antibodies from clarified cell culture supernatant. Multi-column processes provide one way of improving resin capacity utilization, prolong resin lifetime and decrease resin costs; however, it is unclear exactly how many columns should be used. This work compares batch to three different multi-column processes and shows that there is a step change when going from one to two columns but smaller differences among the multi-column processes, and should help in selecting the optimal process for a given production scenario.

Adipose tissue-derived stromal cells (ASC) were differentiated to smooth muscle cells (SMC) and the influence of Poly(1,3-trimethylene carbonate) (PTMC) on the differentiation and function of ASC-derived SMC was assessed. The authors show that ASC-derived SMC are a suitable source for small tissue-engineered blood vessels. Interestingly, mechanical stimulation caused the efficient differentiation of non-differentiated ASC to SMC. This not only renders growth factor stimulation superfluous, but also shortens the time required to engineer vessels.

Oxygen tension can influence ex vivo expansion and physiological function of hematopoietic stem cells (HSCs): Experimental evidence from the current study demonstrated that low oxygen tension favored the expansion of CD34⁺CD38⁻ cells, and CD34⁺ cells expanded under low oxygen tension showed better reconstitution ability in NOD/SCID mice. These results may lay an experimental basis for clinical transplantation of HSCs.

Lipases resistant to methanol are useful catalysts for biofuel production. In this work, the conformational stability of Burkholderia cepacia lipase and its enzymatic activity in transesterification assays is studied. The protein has been shown to present a high robustness towards methanol and good performances in transesterification reactions. These features depict this lipase as a promising enzyme for biofuel industry.

Effective genetic tools would facilitate physiological studies of solventogenic clostridia. In this study, the authors developed CRISPR-based genome editing and expression control systems to achieve successive rounds of gene editing and gene regulation in Clostridium acetobutylicumATCC 824 and Clostridium beijerinckii NCIMB 8052. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia.

The authors are aiming to solve the problem that lack of commercial vaccine against the serogroup B (MenB) of Neisseria meningitidis. This study indicates that transgenic plants could be used as bioreactors to produce a plant-derived fHbp subunit vaccine. And the recombinant fHbp produced from seeds of transgenic plant rHF-22 is a potential candidate for commercial MenB vaccine, which also provides a reference for safe, cheap and large-scale production of plant-made vaccines.

This study aims to establish a novel biosynthetic system for production of important molecule 3-amino-benzoic acid from the simple substrate glucose. The biosynthetic pathway was accommodated in a microbial co-culture composed of two E. coli strains responsible for the upstream and downstream pathway modules, respectively. This approach allowed for rapid identification of the best combination of the constituent E. coli strains and resulted in the production of 48 mg/L 3-amino-benzoic acid.

Efficient hydrolysis of plant polysaccharides is due to the cooperation of multiple enzymes acting on different linkages within the substrate. In this study, new interactions between enzymes are discovered that can help in the design of better enzyme cocktails for plant biomass saccharification. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily-accessible hosts, such as Pichia pastoris, can help in revealing these effects.