Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii

Abstract

The unicellular green alga Chlamydomonas reinhardtii has potential as a cell factory for the production of recombinant proteins and other novel compounds, but mainstream adoption has been hindered by a scarcity of genetic tools and a need to identify products that can be generated in a cost-effective manner. A promising strategy is to use algal chloroplasts as a site for synthesis of high value bioactive compounds such as diterpenoids since these are derived from metabolic building blocks that occur naturally within the organelle. However, synthesis of these complex plant metabolites requires the introduction of membraneassociated enzymes including cytochrome P450 enzymes (P450s). Here, we show that a gene (CYP79A1) encoding a model P450 can be introduced into the C. reinhardtii chloroplast genome using a simple transformation system. The gene is stably expressed and the P450 is efficiently targeted into chloroplast membranes, where it is active and accounts for 0.4% of total cell protein. These results provide proof of concept for the introduction of diterpenoid synthesis pathways into the chloroplast of Chlamydomonas reinhardtii.

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

“…In most cases these are single subunit proteins and therefore involve the introduction of only a single transgene, although there have been a few examples of multigenic engineering of the plastome [32,33]. To date, all of the therapeutic proteins that have been reported are soluble and accumulate in the chloroplast stroma, save for a single report of the targeting of an antibody fragment to the thylakoid lumen [34], although membrane-anchored proteins have been successfully produced in the algal chloroplast [35].…”

Engineering microalgae through chloroplast transformation to produce high‐value industrial products

“…This study concluded that algal biosystems could serve as a platform for a higher and stable expression system of relatively large proteins. Another study in which a plant cytochrome P450 encoding gene from Sorghum bicolor (CYP79A1) was expressed in the chloroplast of C. reinhardtii showed an increased production of diterpenoids (900 ng/mL of the culture) [36]. Wannathong et al [40] used glass beads for the transformation of algal chloroplast and reported that recombinant human growth hormone could be stably expressed up to 25 μg/mL.…”

Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds

“…[19,34] In spite of the multiple examples of favorable effect of chloroplast targeting on heterologous protein accumulation, [34] it was noticed that the optimal subcellular localization might be selected rather empirically, because of individual hardly predictable protein peculiarities. [19] Few recent reports on targeting heterologous CYP450s into chloroplasts, on the one hand, validated their capability to use photosynthetically generated electrons for oxidation reactions; [20,35] on the other hand, indicated drastic dependence of their physiological activity in chloroplasts on molecular structure of transmembrane domain. [36] In the latter work, truncation of the membrane anchor with simultaneous introduction of point mutations into the remaining N-terminal part of CYP720B4 from Picea sitchensis was considered to enable its activity in chloroplasts although strongly reduced transport efficiency.…”

“…DCL was also registered in CYP3A4 extracts ( Figure 3B) together with hydroxylated loratadine derivative exhibiting molecular weight (m/z 399 [MþH]þ, Figure 3E), 16 Da higher than that of loratadine (m/z 383 [MþH]þ, Figure S5A, Supporting Information). Presence of 35 Cl/ 37 Cl atom isotopes in loratadine molecule resulted in %3:1 mixture of protonated molecules in mass www.advancedsciencenews.com www.biotechnology-journal.com spectra and could be regarded as a marker characteristic of loratadine derived metabolites in the extracts. [31] Selective extraction of m/z 399 ion chromatogram allowed detection of traces of four additional loratadine derivatives which were present in similar amounts in both control and experimental samples and are the products of host endogenous enzyme activities ( Figure S6-S8, Supporting Information).…”