Pathway Evolution Through a Bottlenecking-Debottlenecking Strategy and Machine Learning-Aided Flux Balancing.

The evolution of pathway enzymes enhances the biosynthesis of high-value chemicals, crucial for pharmaceutical, and agrochemical applications. However, unpredictable evolutionary landscapes of pathway genes often hinder successful evolution. Here, the presence of complex epistasis is identifued within the representative naringenin biosynthetic pathway enzymes, hampering straightforward directed evolution. Subsequently, a biofoundry-assisted strategy is developed for pathway bottlenecking and debottlenecking, enabling the parallel evolution of all pathway enzymes along a predictable evolutionary trajectory in six weeks. This study then utilizes a machine learning model, ProEnsemble, to further balance the pathway by optimizing the transcription of individual genes. The broad applicability of this strategy is demonstrated by constructing an Escherichia coli chassis with evolved and balanced pathway genes, resulting in 3.65 g L-1 naringenin. The optimized naringenin chassis also demonstrates enhanced production of other flavonoids. This approach can be readily adapted for any given number of enzymes in the specific metabolic pathway, paving the way for automated chassis construction in contemporary biofoundries.

BrpNAC895 and BrpABI449 coregulate the transcription of the afflux-type Cd transporter BrpHMA2 in Brassica parachinensis.

Brassica parachinensis is a popular leafy vegetable. It is able to accumulate high concentration of Cd, however, the molecular mechanism of Cd accumulation is unknown. This study investigated the function and regulatory mechanism of the Cd-responsive metal ion transporter gene BrpHMA2. BrpHMA2 was induced by Cd stress and specifically expressed in vascular tissues, and the protein was localized in the plasma membrane. Heterologous expression of BrpHMA2 enhanced Cd accumulation and Cd sensitivity in transgenic Arabidopsis and yeast. After Cd stress, the transcriptional factors BrpNAC895 and BrpABI449, which may recognize the ABREs in the BrpHMA2 promoter, were also differentially expressed. The transcriptional regulation of BrpHMA2 was further investigated using ChIP-qPCR, EMSA and LUC reporter activity analysis employing the transient expression system of Brassica parachinensis protoplasts and tobacco leaves and the E. coli expression system. By binding to the promoter, BrpNAC895 induced the transcription of BrpHMA2. BrpABI449 might bind to the BrpHMA2 promoter or interact with BrpNAC895 to interfere with the action of BrpNAC895. The findings suggest that BrpHMA2 is a membrane-based afflux-type Cd transporter involved in the Cd2+ uptake and long-distance transport in plants. BrpNAC895 and BrpABI449, which function as the transcription activator and repressor respectively, coregulate BrpHMA2 expression.