Lu Chunzhe, Batianis Christos, Akwafo Edward Ofori, Wijffels Rene H, Martins Dos Santos Vitor A P, Weusthuis Ruud A
Bioprocess Engineering, Wageningen University and Research, Wageningen, The Netherlands.
Laboratory of Systems and Synthetic Biology, Wageningen University and Research, Wageningen, The Netherlands.
Biotechnol Biofuels. 2021 Nov 20;14(1):218. doi: 10.1186/s13068-021-02066-x.
Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols.
AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain.
This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes.
中链长度的α,ω -二醇（mcl -二醇）是聚合物生产中的重要构建单元。最近，利用烷烃单加氧酶系统AlkBGTL和酯化模块Atf1，在大肠杆菌中实现了从烷烃生产微生物mcl -二醇（尽管产量较低）。由于其卓越的多功能性和转化能力，因而具有实现经济可行工艺的潜力，我们评估了工业上稳健的恶臭假单胞菌是否可以成为mcl -二醇的合适生产菌株。
AlkBGTL和Atf1成功表达，这通过烷烃氧化为烷醇以及酯化生成乙酸烷基酯得以证明。然而，转化率低于大肠杆菌，且未完全转化为二醇。通过使用柠檬酸盐而非葡萄糖作为能源，转化率得到提高，这表明碳分解代谢物阻遏发挥了作用。通过过表达AlkBGTL - Atf1的激活剂AlkS以及删除恶臭假单胞菌碳分解代谢物阻遏中的关键基因Crc或CyoB，二乙酰氧基己烷的产量分别提高了76%和65%。去除mRNA的Crc/Hfq结合位点导致二乙酰氧基己烷产量最高。当使用中间产物乙酸己酯作为底物时，检测到了己醇。这表明恶臭假单胞菌表达酯酶，阻碍了相应酯和二酯的积累。对恶臭假单胞菌中存在的16个假定酯酶基因进行了筛选和测试。其中，Est12/K被证明是主要的酯酶基因。删除Est12/K可阻止乙酸己酯和二乙酰氧基己烷的水解。由于解除了分解代谢物阻遏并防止了酯的水解，优化后的菌株从己烷中产生了3.7 mM的乙酸己酯，从乙酸己酯中产生了6.9 mM的6 -羟基己基乙酸酯和二乙酰氧基己烷，与起始菌株相比，产量分别提高了12.7倍和4.2倍。
本研究表明，恶臭假单胞菌的代谢多功能性以及相关碳分解代谢物阻遏会阻碍二醇及相关酯的生产。通过删除主要酯酶可防止在mcl -醇和二醇酯上生长。通过去除Crc/Hfq结合位点可缓解碳分解代谢物阻遏。该策略可用于在假单胞菌及相关物种中高效表达受Crc/Hfq调控的其他基因，以引导生物转化过程。
