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# Cost-effective production of bioplastic polyhydroxybutyrate via introducing heterogeneous constitutive promoter and elevating acetyl-Coenzyme A pool of rapidly growing cyanobacteria - ScienceDirect

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Bioresource Technology
Volume 394, February 2024, 130297
Cost-effective production of bioplastic polyhydroxybutyrate via introducing heterogeneous constitutive promoter and elevating acetyl-Coenzyme A pool of rapidly growing cyanobacteria
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So Young Lee 1
, 
Jeong Seop Lee 1
, Sang Jun Sim
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https://doi.org/10.1016/j.biortech.2023.130297
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Highlights
•
Constitutive promoter Pcpc560 has a high expression level even without any inducer.
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The developed strain expressing phaCAB with Pcpc560 produced 226 mg L−1 of PHB.
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Supplying acetate is more effective than applying stresses in producing PHB.
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Addition of 1 g L−1 of acetate resulted in 607.2 mg L−1 of PHB, a 2.7-fold increase.
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The cost of additive for PHB production could be reduced by more than 300 times.
Abstract
Bioplastic production using cyanobacteria can be an effective strategy to cope with environmental problems caused by using petroleum-based plastics. Synechococcus elongatus UTEX 2973 with heterogeneous phaCAB can produce bioplastic polyhydroxybutyrate (PHB) with a high CO2 uptake rate. For cost-effective production of PHB in S. elongatus UTEX 2973, phaCAB was expressed by the constitutive Pcpc560, resulting in the production of 226 mg/L of PHB by only photoautotrophic cultivation without the addition of inducer. Several culture conditions were applied to increase PHB productivity, and when acetate was supplied at a concentration of 1 g/L as an organic carbon source, productivity significantly increased resulting in 607.2 mg/L of PHB and additive cost reduction of more than 300 times was achieved compared to IPTG. Consequently, these results suggest the possibility of cyanobacteria as an agent that can economically produce PHB and as a solution to the problem of petroleum-based plastics.
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Introduction
Plastic, whose use is steadily increasing due to its low price and convenience, takes a long time to completely decompose after being disposed of, causing severe geophysical and biological impacts worldwide (MacLeod et al., 2021, Zappitelli et al., 2021). Polyhydroxybutyrate (PHB) is a biopolymer belonging to the bioplastic polyhydroxyalkanoate (PHA) class and is attracting attention as a material with similar properties to polypropylene (Price et al., 2020). Bio-derived PHB has biodegradability and biocompatibility, so its use in various areas such as packaging and biomedical fields is being attempted (Yeo et al., 2018). PHB production by photosynthetic microorganisms is characterized by using CO2 as a carbon source through photoautotrophic cultivation (Mastropetros et al., 2022).
One of the photosynthetic microorganisms, cyanobacteria have advantages such as fast growth rate, high photosynthetic rate, and relatively simple genetic manipulation (Quintana et al., 2011). Additionally, producing target substances through mass cultivation of photosynthetic microorganisms can be economically effective (Maroušek et al., 2023a), and among the various CO2 sequestration methods to reduce CO2 in the atmosphere (Maroušek et al., 2023b, Maroušek et al., 2023d), photosynthetic microorganism cultivation is environmentally and economically beneficial method in that it can produce a variety of useful substances, including bioplastics, while avoiding additional CO2 emission (Zhou et al., 2017). Thus, now that global warming is accelerating and climate change appears due to the increase of greenhouse gases, cyanobacteria can be utilized to produce useful substances while reducing CO2 in the atmosphere.
Current studies on PHB production using cyanobacteria are mainly focused on naturally PHB producing species such as Synechocystis sp. PCC 6803 (hereafter PCC 6803) and Synechocystis sp. PCC 6714 that accumulate PHB under stressful environments such as nutrient deprivation (Kamravamanesh et al., 2017, Panda et al., 2006). In addition, genetic engineering including overexpression of native PHB-producing genes (Khetkorn et al., 2016), introduction of PHB synthesis-related genes from other species (Hondo et al., 2015), and overexpression or deletion of genes affecting PHB synthesis (Carpine et al., 2017, Osanai et al., 2014) has been attempted to increase PHB productivity under photoautotrophic condition.
However, there is a fundamental limitation of improving PHB content due to the low biomass productivity of cyanobacteria. To overcome these limitations, phaCAB genes from Cupriavidus necator H16 involved in PHB production was introduced into a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973) in previous study, and phaCAB genes were expressed by inducible trc promoter (Roh et al., 2021). The Ptrc is a type of isopropyl-D-thiogalactopyranoside (IPTG)-inducible promoters from Escherichia coli used for strong gene expression in microorganisms (Brosius et al., 1985). But IPTG is an expensive inducer, and the larger cultivation scale, the more IPTG is required for gene expression, so the overall production cost also increases (Choi et al., 2020). The high production cost and market price compared to petroleum plastics are restrictions to the industrial production and commercialization of PHB (Yeo et al., 2018). The cost of raw materials is a direct factor in determining the plastic production cost resulting in 5 times higher price of bioplastics produced using plants as a source than petroleum-based plastics made from oil, and bioplastics are price competitive only when oil prices are high (Maraveas, 2019, Vochozka et al., 2020). Therefore, a new strategy for more cost-effective production of bioplastic PHB must be introduced.
In this study, the phaCAB genes derived from C. necator H16 was expressed by Pcpc560, a constitutive promoter from cyanobacterium PCC 6803, which does not require the addition of an inducer and enables efficient gene expression in UTEX 2973 (Vasudevan et al., 2019). After gene introduction and mutant development, various cultivation conditions were applied to increase PHB productivity. It was confirmed that adding carbon source was more effective in increasing PHB productivity than applying stress environments such as nutrient deficiency or salt addition. Especially, when acetate, which directly affects the increase in acetyl Coenzyme A (acetyl CoA) pool size which is a raw material for PHB production, was added as a carbon source, the volumetric PHB titer was significantly increase compared to photoautotrophic cultivation and the Synechococcus 2973-phbCAB strain including Ptrc in the previous study. In addition, avoiding an expensive inducer IPTG could significantly reduce the additive cost required to produce the same amount of PHB. This study provides an economical production strategy for cyanobacteria-derived PHB using a constitutive promoter and an organic carbon source that does not require expensive inducer and refined sugar.
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Culture medium and conditions
UTEX 2973 wild type was acquired from University of Texas at Austin (UTEX, USA) and cultivated in BG-11 medium. BG-11 medium consisted of 1.5 g L−1 of NaNO3, 0.036 g L−1 of CaCl2·2H2O, 0.012 g L−1 of ferric ammonium citrate, 0.001 g L−1 of Na2EDTA·2H2O, 0.04 g L−1 of K2HPO4, 0.075 g L−1 of MgSO4·7H2O, 0.02 g L−1 of Na2CO3, and 1 mL L−1 of trace metal solution containing 2.86 g L−1 of H3BO3, 1.81 g L−1 of MnCl2·4H2O, 0.22 g L−1 of ZnSO4·7H2O, 0.39 g L−1 of Na2MoO4·2H2O, 0.079 g L−1 of CuSO4·5H2
Determination of constitutive cpc560 promoter strength in UTEX 2973
For economical PHB production in UTEX 2973 without expensive inducer, constitutive cpc560 promoter from PCC 6803 was selected for phaCAB expression. Before applying to the PHB production, the gfp was introduced into UTEX 2973 as a reporter gene with constitutive Pcpc560 or inducible Ptrc to confirm the strength of each promoter, and UTEX 2973-Pcpc560-gfp and UTEX 2973-Ptrc-gfp strain were obtained (Fig. 1A). UTEX 2973-Ptrc-gfp showed high fluorescence from the 2nd day of cultivation after the
Conclusions
UTEX 2973-Pcpc560-phaCAB developed for cost-effective PHB production was able to produce 226 mg L−1 of PHB through photoautotrophic cultivation and various cultivation strategies were applied to increase PHB productivity. Abiotic stress environments only reduced biomass and did not improve PHB production. Meanwhile, the addition of 1 g L−1 of acetate resulted in 607.2 mg L−1 of PHB production through acetyl-CoA pool size increase, and additive cost reduction of more than 300 times was achieved. 
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Research Foundation of Korea-funded research grant from Korea Carbon to X R&D Center (2020M3H7A1098295), and the Commercializations Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea government (MSIT) (No. 2021B100).
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These authors contributed equally to this work.
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