Zambanini Thiemo, Hosseinpour Tehrani Hamed, Geiser Elena, Sonntag Christiane K, Buescher Joerg M, Meurer Guido, Wierckx Nick, Blank Lars M
iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
BRAIN AG, Darmstädter Straße 34-36, D-64673 Zwingenberg, Germany.
Metab Eng Commun. 2017 Jan 17;4:12-21. doi: 10.1016/j.meteno.2017.01.002. eCollection 2017 Jun.
RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from for use in . Selection markers from that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in . A plasmid was constructed containing the -locus of RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (, ), and two malate transporters (, ) was possible in TZ1 under control of the strong P promoter. Overexpression of , , , and increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L. In bioreactor cultivations of TZ1 P and TZ1 P a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L malate and biomass (73 g L), succinate (20 g L), CO (7 g L), and α-ketoglutarate (8 g L) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.
RK089最近被发现是一种从甘油中高效生产天然苹果酸的菌株。该菌株此前经过了适应性实验室进化，以提高底物摄取率，从而得到了TZ1菌株。培养基优化和工艺参数研究使得该菌株的滴度和产率能够与那些过表达基础代谢途径主要部分的生物体相竞争。如果能够为这种很少使用且相对 obscure 的物种建立基本的遗传工具和方法，代谢工程可能会进一步提高该生物体生产苹果酸的效率。在这里，我们研究并调整了现有的分子工具以便在该物种中使用。来自 confer carboxin、潮霉素、制霉菌素和博来霉素抗性的选择标记可用于该物种。构建了一个包含RK089 -位点的质粒，从而实现了在基因组中的位点特异性整合。使用该质粒，在强P启动子的控制下，丙酮酸羧化酶、两种苹果酸脱氢酶（，）和两种苹果酸转运蛋白（，）在TZ1中得以过表达。在摇瓶中，、、、的过表达使产物（苹果酸）与底物（甘油）的产率提高了54%，滴度高达120 g/L。在TZ1 P和TZ1 P的生物反应器培养中，与参考菌株TZ1相比，生物量形成和甘油摄取率大幅降低，导致比生产率提高了29%（Ssu1）和38%（Mdh2），产率提高了38%（Ssu1）和46%（Mdh2）。产物谱研究显示，以134 g/L苹果酸和生物量（73 g/L）、琥珀酸（20 g/L）、CO（7 g/L）和α -酮戊二酸（8 g/L）作为主要副产物，碳平衡闭合率达到87%。这些结果为进一步优化，特别是组合代谢工程以增加从丙酮酸到苹果酸的通量并减少副产物形成开辟了广泛的可能性。
