Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC

Abstract

Background PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon–neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW). Results We have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic. Conclusion The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

The novel P II -interactor PirC identifies phosphoglycerate mutase as key control point of carbon storage metabolism in cyanobacteria

“…As a proof of principle, we took advantage of the redirection of the carbon metabolism toward PHB synthesis in the PirC-deficient mutant to further increase the levels of PHB in the cell: we engineered the PirC-deficient mutant to express the high processive PHB biosynthetic enzymes from Cupriavidus necator , resulting in a strain that accumulates more than 80% PHB of cell dry mass under nitrogen-depleted conditions. This is by far the highest accumulation of PHB ever reported in a cyanobacterium ( 36 ). This result impressively demonstrates the impact and biotechnological potential of decoupling the regulation by PirC.…”

Cyanobacterium Synechocystis sp. PCC 6803 lacking adc1 gene produces higher polyhydroxybutyrate accumulation under modified nutrients of acetate supplementation and nitrogen-phosphorus starvation

“…Recently, large PHB granules were noted by about 81 %w/DCW PHB under nutrient limiting conditions in a metabolically engineered ΔpirC-REphaAB Synechocystis sp. PCC6803 strain, with heterologous overexpression of Cupriavidus necator phaAB and a disruption of pirC gene encoding phosphoglycerate mutase [23] . The pirC deletion resulted in certain increase of glycogen degradation and lower glycolysis under nitrogen starvation in which PHB accumulation was augmented [ 23 , 29 ].…”

“…PCC6803 strain, with heterologous overexpression of Cupriavidus necator phaAB and a disruption of pirC gene encoding phosphoglycerate mutase [23] . The pirC deletion resulted in certain increase of glycogen degradation and lower glycolysis under nitrogen starvation in which PHB accumulation was augmented [ 23 , 29 ]. On the other hand, we considered that the genetic engineering on any transcription factors may give less impact to induce PHB production whereas the metabolic engineering of competing/neighboring pathways would gain more promising for increasing PHB ( Table 2 ), such as agp knockout [48] , and Xfpk expresstion with double pta_ach deletion [8] .…”

“… [20] The overexpression of Cupriavidus necator phaAB combined with PirC disruption BG 11 containing deprived-N-P and supplemented acetate, phototrophic growth for 20 days 81 %w/DCW Koch et al. [23] Engineered S6803 strain indirectly relating to PHB synthetic pathway The overexpression of native SigE gene encoding RNA polymerase sigma factor BG 11 containing deprived-N, phototrophic growth for 9 days 1.4 %w/DCW Osanai et al. [30] The overexpression of native rre37 gene encoding OmpR-type response regulator BG 11 containing deprived-N, phototrophic growth 1.2 %w/DCW Osanai et al.…”

Про Лідерство України У Виконанні Людством Ноозаповітів В.І. Вернадського На Основі Ноотехнологій І Ноонаук*

“…Він таки розшукав інформацію про перші біологічні пластики й зумів уплинути на Національний Конгрес США так рішуче, що отримав кошти на виробництво упакування для харчових продуктів з Пошуки отримання бактеріальних біопластиків не припиняються і ведуть до нових досягнень, про що свідчить рис. 5, запозичений з публікації [Koch М. et al 2020].…”