Scheel Ryan A, Ho Truong, Kageyama Yuki, Masisak Jessica, McKenney Seamus, Lundgren Benjamin R, Nomura Christopher T
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, NY, United States.
Division of Applied Chemistry, Department of Engineering, Hokkaido University, Sapporo, Japan.
Front Bioeng Biotechnol. 2021 Feb 26;9:618259. doi: 10.3389/fbioe.2021.618259. eCollection 2021.
Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by β-oxidation deficient recombinant LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L, 0.42 g L h), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.
生产具有严格定义组成的中链长度聚（3-羟基链烷酸酯）[PHA]聚合物是一个重要的研究领域，有助于扩大这些有前景的生物基和可生物降解材料的应用范围并改善其性能。具有均聚物或特定组成的PHA聚合物表现出有吸引力的材料特性，例如相对于聚（3-羟基丁酸酯）[PHB]具有更高的柔韧性和弹性；然而，这些聚合物在天然产生PHA的生物体中难以进行生物合成，并且在开发高密度培养方法同时保持组成控制方面的研究较少。在本研究中，我们在搅拌罐反应器中开发并优化了补料分批发酵工艺，首先通过β-氧化缺陷型重组LSBJ以葡萄糖作为仅用于生长的共底物，从癸酸生物合成聚（3-羟基癸酸酯）[PHD]。细菌分两个阶段培养，一个是生物质积累阶段（37°C，pH 7.0），以葡萄糖作为主要碳源，另一个是PHA生物合成阶段（30°C，pH 8.0），同时补料葡萄糖和脂肪酸。通过对半定义培养基组成和葡萄糖进料速率的迭代优化，6.0 g癸酸转化为PHD，摩尔产率为87.5%（4.54 g/L）。发酵过程中逐步增加癸酸进料量与PHD产量增加相关，最终25 g癸酸进料转化为PHD，产率为89.4%（20.1 g/L，0.42 g/(L·h)），此时泡沫变得无法控制。己酸, 辛酸, 10-十一碳烯酸和10-溴癸酸均各自以20 g添加，并成功聚合，产率范围为66.8%至99.0%（9.24至18.2 g/L）。使用这种生物反应器策略，辛酸/癸酸和辛酸/10-叠氮基癸酸（各8:2摩尔比）的共脂肪酸进料以几乎相同的比例和高产率产生了它们各自的共聚物，表明这些方法可用于控制PHA共聚物组成。
