Metabolic engineering of the cyanobacterium Synechocystis sp. PCC 6803 for the production of astaxanthin

Abstract

Synechocystis sp. PCC 6803 is a photosynthetic eubacterium capable of using light energy to generate biomass from atmospheric CO2 and is considered to be the model organism of photosynthetic microbes. Much of the knowledge accumulation related to this organism has centered on the cellular photosynthetic process because this organism has many similarities to the chloroplasts of higher order plants. Synechocystis also shows great promise as a microbial cell factory, as scientific studies describing metabolite production from this organism continue to accumulate in the literature. While these studies highlight the considerable amount of gains made in regards to production in Synechocystis, they also shed light on the considerable amount of gaps in knowledge regarding many aspects of this organism. As the field of metabolic engineering continues to grow within Synechocystis, researchers must continue to develop production pathways that leverage comprehensive engineering strategies that help in shedding light on critical engineering hurdles. This information is critical for the successful development of photosynthetic microbes as cellular production platforms capable of generating titers similar to those seen in other cellular systems utilized to generate economically viable metabolites for humankind. In this work, we utilized several metabolic engineering strategies to manipulate the carotenoid biosynthesis pathway in Synechocystis for the production of the non-native carotenoids, astaxanthin as well as canthaxanthin. A Synechocystis mutant was engineered with an insertion of a β-carotene di-ketolase gene crtW148 from Nostoc punctiforme, insertion of an additional copy of the endogenous β-carotene hydroxylase gene crtR from Synechocystis, and an open reading frame disruption of the endogenous β-carotene mono-ketolase gene crtO. These manipulations generated a mutant capable of an increase in the overall carotenoid content by 178 ± 10% % of that seen in wild type cells as well as astaxanthin titers that reached production rates of 1.11 ± 0.07 mg/l/day and canthaxanthin titers reaching 1.49 ± 0.05 mg/l/day. To add upon this work, we leveraged several promoters, the PSCA6-2 promoter as well as the PsigA promoter to control the expression of the crtW148 gene within several constructs. These promoters were generated in a research study we performed that leveraged rational design strategies to develop a suite of promoters capable of driving gene expression as various strengths within Synechocystis. This study generated a library of 10 promoter-constructs capable of a dynamic range of expression strength, exhibiting a 78 fold change between the lowest expressing promoter, Psca8-2 and the highest expressing promoter, Psca3-2 when tested within Synechocystis. Use of the PSCA6-2 promoter within the carotenoid pathway engineering experiment increased carotenoid production of target carotenoids by 150% to 197% over production seen from the same constructs run by the promoter PsigA. In addition to engineering of the carotenoid biosynthesis pathway, we also tested the impacts of diel cycle light conditions on carotenoid production and accumulation. When exposed to 12 hour light/dark conditions, the mutant crtR::cruB::ΔcrtO-PSCA6-2::crtW generates carotenoids at rates of 43 ± 14.8 % of that of the same culture grown in constant light conditions. We hypothesized that this lag was caused by the endogenous cellular control of the carotenoid pathway initiated by the metabolic burden placed on the cell. We also hypothesize that this metabolic burden was caused by the engineered constitutive expression of the astaxanthin producing genes during dark conditions. To address potential concerns of constitutive expression of pathway genes during stress conditions like the dark conditions highlighted in the astaxanthin work, our lab constructed a chemically inducible construct for use in Synechocystis that is based on the tac repressor. Upon chemical induction with IPTG, this same mutant strain was capable of exhibiting an average 24X increase in GFP expression over that of the repressed state. In addition to this work, we studied several light induced promoters to better understand their ability to control gene expression during various light conditions in neutral locations within the Synechocystis genome. We identified that the PpsbAII promoter functions very differently in light and dark conditions when it is moved from its native location within the genome. As many researchers utilize this promoter to control gene expression, this information may be critical to fully understanding gene expression of pathways leveraging this promoter construct. Three additional promoter constructs, the PpsbAIII. PgroEL2, and PsigD promoters were also tested for differential expression in light and dark conditions within the neutral region slr0168. Additionally, nucleotide mutations were made to regions within the PpsbAII promoter, to better understand this promoter’s sensitivity to varying light intensities.