Gottlieb Katrin, Albermann Christoph, Sprenger Georg A
Microb Cell Fact. 2014 Jul 11;13(1):96. doi: 10.1186/s12934-014-0096-1.
For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported.
L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM C(Phe)/C(Glucose) and a maximal space-time-yield (STY) of 0.13 g l(-1) h(-1) was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM C(Phe)/C(Glycerol)), but the maximal STY was higher (0.21 g l(-1) h(-1)). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpK(G232D)) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM C(Phe)/C(Glycerol); 0.25 g l(-1) h(-1) and 0.21 mM C(Phe)/C(Glycerol); 0.23 g l(-1) h(-1), respectively), the combination of extra genes of glpX and tktA together led to an increase in maximal STY of about 80% (0.37 g l(-1) h(-1)) and a carbon recovery of 0.26 mM C(Phe)/C(Glycerol).
Enhancing the gene copy numbers for glpX and tktA increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in E. coli has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P.
要生产L-苯丙氨酸（L-Phe），需要两分子磷酸烯醇丙酮酸（PEP）和一分子赤藓糖-4-磷酸（E4P）。PEP来源于糖酵解，而E4P是在磷酸戊糖途径（PPP）中形成的。常用于通过重组大肠杆菌生产L-Phe的葡萄糖，是通过依赖PEP的磷酸转移酶系统摄取的，该系统可提供6-磷酸葡萄糖（G6P）。G6P可进入糖酵解或PPP。相比之下，甘油由依赖ATP的甘油激酶（GlpK）磷酸化，从而节省一分子PEP。然而，生长和提供E4P需要两个糖异生反应（果糖-1,6-二磷酸醛缩酶、果糖-1,6-二磷酸酶，FBPase）。甘油已成为生物技术的重要碳源，并且有关于从甘油生产L-Phe的报道。然而，FBPase和转酮醇酶反应对L-Phe生产的影响尚未见报道。
比较了亲本菌株FUS4/pF81（编码aroF、aroB、aroL、pheA的质粒编码基因）在葡萄糖和甘油作为碳源时的L-Phe生产率。在葡萄糖上，发现最大碳回收率为0.19 mM C（Phe）/C（葡萄糖），最大时空产率（STY）为0.13 g l⁻¹ h⁻¹。使用甘油时，最大碳回收率几乎相同（0.18 mM C（Phe）/C（甘油）），但最大STY更高（0.21 g l⁻¹ h⁻¹）。我们分别提高了染色体基因glpK（编码甘油激酶）、tktA（编码转酮醇酶）和glpX（编码果糖-1,6-二磷酸酶）的基因拷贝数。glpK（或其抗反馈变体glpK（G232D））的过表达对生长速率影响很小；L-Phe产量比FUS4/pF81低约30%。而glpX或tktA的过表达对生产率影响较小（分别为0.20 mM C（Phe）/C（甘油）；0.25 g l⁻¹ h⁻¹和0.21 mM C（Phe）/C（甘油）；0.23 g l⁻¹ h⁻¹），glpX和tktA的额外基因组合在一起使最大STY提高了约80%（0.37 g l⁻¹ h⁻¹），碳回收率为0.26 mM C（Phe）/C（甘油）。
增加glpX和tktA的基因拷贝数可提高从甘油生产L-Phe的生产率，而不影响生长速率。在大肠杆菌中对甘油代谢进行工程改造以生产L-Phe时，必须平衡糖异生、糖酵解和PPP途径，以改善前体PEP和E4P的供应。
