Rational Mutagenesis to Enable Yeast DNA Polymerase ? (POL3) Tobecome a Calcium-Sensitive Recording Device

 Claudia Durall^1*, Pia Lindberg¹ and Peter Lindblad¹

¹Chemistry Ångström, Uppsala Univeristy, Uppsala, Sweden

claudia.durall@kemi.uu.se

It is possible to genetically engineer cyanobacteria to produce a variety of biofuels [1-3]. However, the amount of the selected product is low. It has been demonstrated that increased carbon fixation can increment the production of carbon based biofuels and other products of interest in photosynthetic organisms.

In addition to RuBisCO, Phosphoenolpyruvate carboxylase (PEPc) may also fix carbon in cyanobacteria [4]. I have designed three different engineered strains by overexpressing the native pepc in the cyanobacterium Synechocystis PCC 6803 and I created cells with one (WT+PEPc) or two additional copies of pepc (WT+2xPEPc), as well as cells with additional copies of pepc, ppsa and mdh (WT+PPSA+PEPc+MDH). The additional copies were designed to replace the psbA2 gene in the genome. However, since the additional copy of pepc is identical to the native, single recombination with the native pepc occurred in the engineered cells. SDS-PAGE/Immunoblot demonstrated that more PEPc protein is present in the engineered cells compared to in wild type cells with an incremented level of PEPc with increasing the number of pepc. Interestingly, WT+2xPEPc, and subsequent higher PEPc protein level and higher in vitro PEPc activity, grow faster than the control strain, but only under conditions of very low light intensity [5].

According to our experience, heterologous, compared to homologous, expression has shown better results in terms of protein level and activity. Thus, the pepc from Synechococcus PCC 7002 and 7942 was introduced in the cyanobacterium Synechocystis PCC 6803 either in the chromosome or in a self replicative vector. Interestingly, when a single copy of pepc from either Synechococcus strain was introduced into the chromosome, the in vitro PEPc activity was similar to WT+2xPEPc. Differently, only when the pepc from Synechococcus PCC 7942 was expressed in a self replicative vector, the in vitro PEPc activity was similar to the heterologous expression of pepc from either Synechococcus in the chromosome or to WT+2xPEPc. Currently, the PEPc protein level and the growth of the engineered strains are being examined.

References

[1] S.A. Angermayr, K.J. Hellingwerf, P. Lindblad, M.J. Teixeira de Mattos. Energy biotechnology with cyanobacteria. Curr Opin Biotechnol 20 (3) (2009) 257-263.

[2] P. Lindblad, P. Lindberg, P. Oliveira, K. Stensjö, T. Heidorn. Design, engineering, and construction of photosynthetic microbial cell factories for renewable solar fuel production. Ambio 41 (2) (2012) 163–168.

[3] R. Miao, A. Wegelius, C. Durall, F. Liang, N. Khanna, P. Lindblad. Engineering cyanobacteria for biofuel production. In: Modern Topics in the Phototrophic Prokaryotes, Environmental and Applied Aspects (2017) Chapter 11, (in press). ISBN: 978-3-319-46259-2.

[4] C. Durall, P. Lindblad. Mechanisms of carbon fixation and engineering for increased carbon fixation in cyanobacteria. Algal Res 11 (2015) 263-270.

[5] C. Durall, N. Rukminasari and P. Lindblad. Enhanced growth at low light intensity in the cyanobacterium Synechocystis PCC 6803 by overexpressing phosphoenolpyruvate carboxylase. Algal Res 16 (2016) 275-281.