A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in E. coli.

Human neurturin (NTN) is a cystine knot growth factor with potential therapeutic use in diseases such as Parkinson's and diabetes. Scalable high titer production of native NTN is particularly challenging because of the cystine knot structure which consists of an embedded ring comprised of at least three disulfide bonds. We sought to pursue enhanced scalable production of NTN in Escherichia coli. Our initial efforts focused on codon optimization of the first two codons following AUG, but these studies resulted in only a marginal increase in NTN expression. Therefore, we pursued an alternative strategy of using a bicistronic vector for NTN expression designed to reduce mRNA secondary structure to achieve increased ribosome binding and re-initiation. The first cistron was designed to prevent sequestration of the translation initiation region in a secondary conformation. The second cistron, which contained the NTN coding sequence itself, was engineered to disrupt double bonded base pairs and destabilize the secondary structure for ribosome re-initiation. The ensemble approach of reducing NTN's mRNA secondary structure and using the bicistronic vector had an additive effect resulting in significantly increased NTN expression. Our strain selection studies were conducted in a miniaturized bioreactor. An optimized strain was selected and scaled up to a 100 L fermentor, which yielded an inclusion body titer of 2 g/L. The inclusion bodies were refolded to yield active NTN. We believe that our strategy is applicable to other candidate proteins that are difficult-to-express due to stable mRNA secondary structures. Biotechnol. Bioeng. 2017;114: 1753-1761. © 2017 Wiley Periodicals, Inc.

Characterization of oxygen transfer in a 24-well microbioreactor system and potential respirometric applications.

The assessment of microbial processes is often done in Microbioreactor systems (MBRs), which allow for parallel cultivation in multiple independent wells. MBRs often include dissolved oxygen sensors, which are convenient for process characterization through oxygen uptake rate and other respirometric determinations. In order to assess respirometric potential of MBRs, a complete assessment of the DO fluorescent quenching sensors was done, showing that they presented a typical error of 0.56%, a signal to noise ratio of 189, a response time from 5.7 to 7.2 s and no drift over a period of 24 h. Then, KLa in the MBR was measured with different cassette and cap designs, liquid volumes, agitation rates, gas flow rates, temperatures and ionic strengths. KLa ranged from 8 to 90 h(-1), with a standard deviation between replicates from 2.8 to 17.5%. From these results and a numerical simulation, it was shown that the MBR tested allow the determination of oxygen uptake rates in a range from 0.038 to 3390 mg L(-1) h(-1), with a determination error less than 15%. Besides OUR determination, it was concluded that the MBR tested is also a convenient tool for dynamic pulse respirometry methods, based on experimental confirmation with four different cultures.